Peptides and methods for treatment of neurodegenerative diseases

ABSTRACT

The present disclosure provides pharmaceutical compositions comprising rationally designed peptide analogs of the p65-TAD binding region of GILZ to selectively sequester activated p65. Structural and functional analyses suggest that select GILZ analog (GA) bind p65-TAD with optimum affinity, exhibit an estimated half minimal lethal dose comparable to known peptide drugs and suppress Aβ1-42 induced cytotoxicity. Furthermore, the present disclosure provides uses and methods of using the pharmaceutical compositions, and uses and methods of using pharmaceutical formulations comprising the pharmaceutical compositions, for the treatment of neurodegenerative diseases such as Alzheimer&#39;s Disease, Parkinson&#39;s Disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT InternationalApplication Serial No. PCT/US2017/018645, filed on Feb. 21, 2017, andclaims the benefit under 35 USC § 119(e) of U.S. Provisional ApplicationSer. No. 62/298,216, filed on Feb. 22, 2016, the entire disclosures ofwhich are incorporated herein by reference

GOVERNMENT RIGHTS

This invention was made with government support under TR000006 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 13, 2018, isnamed 281227 ST25.txt and is 16.3 kilobytes in size.

BACKGROUND

The burden of neurodegenerative pathologies including Parkinson's andAlzheimer's disease (AD) is increasing exponentially worldwide. Anaccumulating body of evidence suggests that a combination of age relatedchanges in the central nervous system (CNS) with excessive or prolongedinflammatory responses contribute to the pathophysiology ofneurodegeneration, synaptic dysfunction and hippocampal behaviordeficits in neurodegenerative diseases. In the United States, anestimated 5.1 million Americans currently have AD. The number isprojected to increase by 40%, affecting 7.1 million individuals over 65years of age, by 2025. Translated into healthcare costs, currently thedirect cost of caring for AD is estimated at $226 billion and isprojected to reach $1.1 trillion by 2050. Thus, there is an urgent needto grow the therapeutic pipeline for AD with agents capable of slowingthe disease progression.

The pleiotropic transcription factor, nuclear factor-kappa B (NF-κβ) isinduced by many physiological and pathological stimuli in the CNS. TheNF-κβ family consists of five members, p50, c-rel, p65, RelB and p52that can diversely combine to form transcriptionally active dimers. Ithas been suggested that the nature of the dimers determine the effectsof activated NF-κβ. While c-rel containing dimers preferentially promotetransactivation of anti-apoptotic factors, activation of p65/p50 dimersprimarily enhance inflammatory and pro-apoptotic gene transcription.Positive and negative regulatory mechanisms maintain a balance betweenthe neuroprotective c-rel dimers and the predominantly deleteriousp65:p50 dimers in healthy CNS.

In AD, secondary stimuli such as accumulating beta amyloid and oxidativestress increase activation of p65:p50 dimers in glial cells. Theextracellular amyloid plaques mainly include the 42-residue long Aβamyloid b-peptide (Aβ), obtained by proteolytic cleavage from the muchlarger amyloid precursor protein (APP). APP is thought to play a role incellular adhesion and motility. In the amyloidogenic pathway, APP isfirst cleaved by the β-secretase at the amino-terminus generatingsoluble sAPPβ and a carboxy-terminal fragment, which is then cleaved byy-secretase producing the Aβ₁₋₄₂ peptides. Cleavage of APP byβ-secretase (also known as beta site amyloid precursor protein cleavingenzyme-1 (BACE-1)) is considered to be the rate limiting step for Aβgeneration.

The promoter region of human BACE-1 gene exhibits κB binding elementsthat physically interact with NF-κβ p65. Activation of NF-κβ p65increases endogenous BACE-1 transcription and consequent Aβ production.Increased presence of activated p65 and BACE-1 has been observed aroundAβ plaques in postmortem AD tissues. Extracellular Aβ peptidespredominantly activate p65:p50 dimers in glia and post-mitotic neuronsand enhance transactivation of inflammatory and pro-apoptotic genes.Increased presence of IL-1β, IL-6, and TNF-α have been reported in theaffected tissues, serum and CSF of AD patients. Elevated Bax(proapoptotic) to Bcl-2 (anti-apoptotic) ratio have been observed in Aβstimulated neuronal cells. A feed-back loop of excessive Aβaccumulation, NF-κβ activation, cytotoxicity and more Aβ productionculminate in neurodegeneration. Conditional knock out of p65 has beenshown to attenuate BACE-1 transcription and Aβ genesis in AD mice.Absence of p65 co-factors such as p300/CREB binding associated factorhas been shown to mediate resistance to Aβ induced toxicity. Thus,although neuronal p65 has been shown to contribute to the physiologicalfunctions of synapse formation and transmission, considerable evidencesuggest that excessive activated p65 in the CNS lead toneurodegenerative pathology. Hence selective inhibition of activated p65could ameliorate pathologies wherein inflammation is closely associatedwith degeneration, such as AD.

Moreover, spinal cord injury (SCI) is a devastating affliction thataffects hundreds of thousands of U.S. citizens alone with approximately15,000 more diagnosed each year. Mechanical trauma to the spinal cordtriggers a sequence of secondary pathological events includingwidespread cell death, axonal disruption, and inflammation ultimatelyleading to various severities of functional deficits. In addition,multiple reports suggest that the SCI patients frequently exhibitlong-term cognitive impairments. Although very often discounted asprobably reflecting concurrent brain injury, recent preclinical studiesindicate that SCI induces chronic brain inflammation that culminates inneurodegeneration and cognitive decline. However, few studies haveattempted to evaluate these associations at a mechanistic level andlimit such deficits or promoting recovery.

Multiple mechanisms linking chronic SCI with neurodegeneration andcognitive decline have been proposed, such as potential autoimmuneresponses, loss of immune tolerance to amyloid beta andneuroinflammation. Stereological quantification and immunohistochemicalstudies have shown that the microglia in the hippocampus and cortex ofmice at day 8 post-SCI exhibit an activated phenotype with elevatedIba1, chemokine-ligand 21, inflammatory cytokines and several cell cyclerelated genes. Increased proliferation of activated microglia has beenobserved even 12 weeks after SCI. It has been observed that themicroglia were increased in the hippocampus at day 7 and day 14 postSCI. Furthermore, preliminary data suggest that the inflammatorycytokines are elevated in the brain homogenates harvested at day 7post-SCI.

Although generally the blood brain barrier protects the brain againstpathogens, activation of microglia can be mediated by several dangerassociated molecular pattern (DAMP)s or auto-antigens such as MBP andamyloid beta. High-Mobility Group Box 1 (HMGB1) is a prototypical DAMPshown to be upregulated in the spinal cord homogenates and in thecirculation as early as day 3 post-SCI and persist at elevated levelsfor extended period even up to 2 years. HMBG1 binds two cognizantreceptors, toll like receptor-4 (TLR4) and receptor for advancedglycation end (RAGE) products. Furthermore, HMBG1 has been shown toupregulate the microglial cell responses to lipopolysaccharides. Takentogether, it was hypothesized that HMBG1 will initiate hippocampalmicroglia via TLR4 signaling with subsequent upregulation of MHC classII and the costimulatory receptors CD80 and CD86 priming the cells forantigen (auto-antigen) presentation.

The critical role of inflammation in the persistence of traumatic SCIand the significant contribution of the inflammatory response inincreasing the potential risk of damage at distant intracranial sitessuggest early control of inflammation could not only enhance tissuerepair at the site of injury but also decrease the chances for extendedCNS damage. NF-κβ is a major regulator of inflammatory responses.Aberrant activation of NF-κβ p65 has been observed in both glial cellsand neurons in SCI, which in turn leads to increased transactivation ofproinflammatory cytokines and pro-apoptotic factors. Therefore,targeting NF-κβ has been recognized as an effective therapeutic strategyfor SCI13. The profound therapeutic efficacy of glucocorticoids (GC),widely prescribed as a first line therapy for SCI is largely attributedto the inhibition of NF-κβ by GC receptor in the nucleus. However,binding of GC receptor to specific negative response DNA elementsinduces cis-repression of osteocalcin and other target genes thatprecipitate many side-effects of GC including osteoporosis, diabetes andobesity. Strategies that duplicate the beneficial effects of GC andinhibit NF-κβ transactivation are likely to be of significanttherapeutic advantage for SCI. As such, suppressing activated NF-κβcould be utilized to reduce tissue damage in patients post-SCI. Theefficacy of glucocorticoids in acute SCI, as well as of that of many ofcompounds such butein, curcumin or tamoxifen evaluated in preclinicalmodels of SCI have been attributed indirectly to NF-κβ inhibition.

Structurally, p65 has an amino terminal rel homology domain (RHD), anuclear localization sequence (NLS) masked by the IκB inhibitory complexand a carboxy terminal transactivation domain (TAD). The transactivationactivity of p65 is mediated by interactions of the TAD withco-regulators and the basal transcription machinery. Glucocorticoidinduced leucine zipper (GILZ) is a p65 binding protein that sequestersactivated p65 and inhibits transactivation of inflammatory and apoptoticfactors. Mutational and binding analyses localized the interactioninterface to the proline rich carboxy terminus of GILZ and the TAD ofp65. Molecular modeling suggested that the p65 binding domain of GILZadopts a flexible polyproline type II (PP_(II)) helical conformationthat interacts with the highly conserved F⁵³⁴/F⁵⁴² in p65-TAD.

In recent years, considerable success has been achieved in thedevelopment of structurally engineered peptide analogs of the bindingepitope(s) of a protein as therapeutic leads. The strategy isincreasingly adopted in the design of mimics of proline rich motif thatmediate transient intermolecular interactions. The specificity of theinteraction is determined by the nature of the proline rich bindingdomain interface.

Thus, there exists a need for novel pharmaceutical compositions andpharmaceutical formulations for the treatment of neurodegenerativediseases and for suppressing neuroinflammation in disease states such asSCI.

The present disclosure provides pharmaceutical compositions comprisingrationally designed peptide analogs of the p65-TAD binding region ofGILZ to selectively sequester activated p65. Structural and functionalanalyses suggest that select GILZ analog (GA) bind p65-TAD with optimumaffinity, exhibit an estimated half minimal lethal dose comparable toknown peptide drugs and suppress Aβ1-42 induced cytotoxicity.Furthermore, the present disclosure provides uses and methods of usingthe pharmaceutical compositions, and uses and methods of usingpharmaceutical formulations comprising the pharmaceutical compositions,for the treatment of neurodegenerative diseases such as Alzheimer'sDisease, Parkinson's Disease, multiple sclerosis, and amyotrophiclateral sclerosis (ALS). Moreover, the present disclosure provides usesand methods of using the pharmaceutical compositions, and uses andmethods of using pharmaceutical formulations comprising thepharmaceutical compositions, for the treatment of a spinal cord injury,for example by reducing inflammation in the spinal cord of a patient orin the brain of a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show comparative modeling of GILZ analogs. Superimpositionof human delta sleep inducing peptide (DSIP; PDB: 1DIP) with thepredicted model of human GILZ (FIG. 1A), superimposition of indicatedanalog (blue) with the critical residues in the proline glutamic acidrich region of human GILZ model (red) (FIGS. 1B-1F). Structuralsimilarity in terms of root mean square deviation (RMSD) for eachsuperimposition is indicated.

FIGS. 2A-2F show the docked complex of wild type GILZ-PER (prolineglutamic acid rich region) or GILZ analog (GA) with human p65-TAD.Representative molecular model of p65-TAD docked (blue) with wild typeGILZ-PER (FIG. 2A) and indicated analog (FIG. 2B-2F) (red) are shown.The residues in each analog <5 Å distance of highly conserved residuesin p65-TAD2 (Phe or Phe) and the “LXXLL” motif in p65-TAD₁ that suggestproximity with residues critical for transcriptional activity are shown.

FIGS. 3A-3F show GA-rp65 binding analysis. Binding between theplate-bound GILZ analog (GA) or control peptide (CP) (20 μM-640 μM) atincreasing concentration and the r-p65 was detected with the anti-DDK asdescribed in the methods section. A dose dependent decrease in percentbound r-p65 was observed in association with the GA (FIG. 3A). Scatchardplot analysis of bound p65 (A₀−A)/(A₀) against the ratio of bound p65 tofree GA (y=(A₀−A)/(A₀)/[(A₀−A)/(A₀)/(a₀−i₀)] was used to determine thedissociation constant for the interaction between indicated GA and r-p65(FIG. 3B-3F).

FIGS. 4A and 4B show the effect of GILZ analog (GA) on cellularmorphology and metabolic activity in neuroblasts. Differentiated SK-N-SHneuroblastoma cultures were exposed to individual GA or control peptide(CP) at the indicated concentration for 24 hours. (FIG. 4A). Phasecontrast imaging of the cells shows no apparent adverse effects onmorphology of cells exposed to GA-1 or GA-2 or CP-1 or CP-2 at eitherconcentration. Exposure to CP-3 at both concentrations showedmorphological changes consistent with cell death. (FIG. 4B). Celllysates were assessed by CTG assay to determine relative levels ofintracellular ATP. An increase in Relative Luminescence Units (RLU)suggesting cellular viability was observed with all treatments exceptCP-3.

FIGS. 5A-5G show the effect of GA on lactate dehydrogenase (LDH)release. U373 MG astroglioma cells were exposed to increasingconcentrations of indicated GILZ analog (GA) or control peptide (CP)(0.5 μM to 1 mM) for 24 hours. The release of LDH into the cell culturesupernatant from damaged cells was measured. Percentage of LDH wascalculated as the ratio of the difference between the peptide treatedand untreated cells to that of the difference between the positivecontrol and the untreated cells (FIG. 5A). The IC50 was determined bylogarithmic extrapolation (FIG. 5C-5G). Flow cytometric analysis ofAnnexin positive U373 cells treated as indicated was determined as ameasure of apoptosis (FIG. 5B).

FIGS. 6A-6E show the effect of GILZ analog (GA) treatment on human fetalbrain cells. Primary cultures of HFB (Div 17) were exposed to Aβ1-42 andtreated with 50 μM of indicated GA or control peptide (CP). Cytoplasmicextracts of cells collected at 24 hours was assessed for viability byCTG assay. Data are presented as ARLU (difference in relativeluminescent units (RLU) between the Aβ1-42 exposed cells and unexposedcells) (FIG. 6A, FIG. 6B). Culture medium collected at 24 hrs wasassessed for indicated cytokines (FIG. 6C, FIG. 6D). Effect of GA onNF-κβ activation. Primary cultures of HFB exposed to Aβ1-42 (10 μg/ml)and treated with indicated GA or CP as above were harvested at the endof 4 hours. 5 μg of nuclear extract was tested for binding of theactivated p65 NF-κβ subunit to an NF-κβ consensus sequence using theTrans AM NF-κβ ELISA kit. The p65 DNA binding activity was calculated asthe ratio of absorbance from Aβ1-42 stimulated cells to that ofunstimulated cells (FIG. 6E). Values are the average/−S.D. *=p<0.05 ascompared to Aβ1-42 exposed cells, @=p<0.05 as compared with Aβ1-42 andCP-1 or CP-2 treated cultures

FIG. 7 shows the Schematic representation of pathological mechanisms ofAD and points of intervention by GA. Increased oxidative stress andother age related changes upregulate NF-κβ p65 which in turn increasetranscription of amyloid precursor protein (APP) and/or beta siteamyloid precursor protein cleaving enzyme-1 (BACE-1) leading togeneration and accumulation of Aβ peptides in the CNS parenchyma. Glialcells exposed to Aβ peptides exhibit increased p65 activation andsecrete inflammatory and apoptosis mediators. Affected neuronsupregulate Aβ peptides and the vicious cycle of Aβ deposition,inflammation and neuronal apoptosis leads to AD. GILZ analogs (GA) byvirtue of binding activated NF-κB p65 blocks Aβ generation andsuppressing inflammation, thereby ameliorating AD pathology.

FIGS. 8A-8D show comparative modeling and docking of PGA:p65 (FIG.8A-FIG. 8B). Superimposition of indicated PGA (blue) with the criticalproline rich region of human GILZ (red), both built using PDB:1DIP astemplate. Root mean square deviation (RMSD) representing structuralsimilarity is given. (FIG. 8C-FIG. 8D). Representative model of p65-TAD(blue) docked with indicated PGA (red). The p65-TAD residues criticalfor transcriptional activity at close proximity (RMSD <5 Å) to PGA arehighlighted.

FIGS. 9A-9C show binding between plate-bound PGA (20{circumflex over( )}M-640{circumflex over ( )}M) and r-p65 (15 pg/ml). (FIG. 9A) All PGAexhibit a dose dependent decrease in % bound r-p65. (FIGS. 9B-9C).Scatchard plot analysis of bound p65 against the ratio of bound p65 tofree PGA-4 (representative) to determine KD. A:absorbance of PGA:p65,Ao: absorbance of r-p65-DDKanti-DDK in the absence of bound PGA, ao:total PGA concentration and io: total r-p65 concentration.

FIGS. 10A and 10B show U373 cells were exposed to increasingconcentrations of different PGA or wild type GILZ-P (0.5 μM to 500 μM)for 24 hours. Percent LDH released into the medium was calculated as theratio of the difference in PGA treated to untreated cells to that of thedifference between the positive control and untreated cells (FIG. 10A).The IC50 was determined by logarithmic extrapolation (FIG. 10B).

FIGS. 11A-11D show primary cultures of HFB pretreated with indicated PGAat IC50 concentration was exposed to vehicle or 10 μM of Aβ₁₋₄₂. Cellviability at 24 hours was assessed by CTG assay. Data are presented asΔRLT (difference in relative luminescent units) between Aβ1-42 exposedand unexposed cells) (FIG. 11A, FIG. 11B). Culture medium was assessedfor IL-6 (FIG. 11C). HFB cultured similarly was harvested at the end of4 hours (FIG. 11D). Activated p65 in nuclear extract was tested forbinding an NF-κB consensus sequence. The p65 DNA binding activity wascalculated as the ratio of absorbance from Aβ₁₋₄₂ stimulated cells tothat of unstimulated cells. Data are mean/−S.D. *=p<0.05.

FIG. 12 shows a weighted rank order based on parameters of known peptidedrugs suggested that the PGA-2 and PGA-4 exhibit significant drug likeproperties.

FIGS. 13A-13E show HFNC cultures at DIV20. A phase contrast image (FIG.13A) and nuclear staining with DAPI (FIG. 13B) are displayed. In FIGS.13C, 13D, and 13E, cells labelled with indicated antibody and mergedimage. Pan-neuronal antibody, a cocktail of mAbs, identifies somatic,nuclear, dendritic and axonal proteins distributed across the panneuronal architecture. Significant individual and co-labeling withpan-neuronal and anti-GFAP mAb suggests presence of immature neural stemcells as well as both differentiated neurons and astrocytes. Arrows andarrowheads point to cells labeled only with pan-neuronal mixture oranti-GFAP respectively.

FIGS. 14A-14D show the docked complex of GA-1 with human p65-TAD.Representative molecular model of p65-TAD docked with GA-1 (FIG. 14A) orGA-2 (FIG. 14B). The critical proline in each GA and the criticalphenylalanine are highlighted in red and blue respectively. The distancebetween the CP atoms of the two residues in the interface is shown. Cand D show Ramachandran plot of GA-1 (FIG. 14C) or GA-2 (FIG. 14D) withthe phi and psi angles of the critical proline (in red) suggestive ofPP_(II) helical conformation.

FIGS. 15A-15H show representative images of degrees of microgliosis inmouse hippocampus. FIG. 15A-15G shows representative IHC section stainedfor Iba+ microglia in hippocampus of representative mouse treated withthe indicated compound (FIG. 15H) shows the mean optical density of the3,3′-diaminobenzidine (DAB)-positive cells depicting microglia in groupsof vehicle or indicated compound treated mice.

FIGS. 16A-16H show representative images of degrees of astrogliosis inmouse hippocampus. FIG. 16A-16G Shows representative IHC section stainedfor GFAP+ microglia in hippocampus of representative mouse treated withthe indicated compound (FIG. 16H) shows the mean optical density of the3,3′-diaminobenzidine (DAB)-positive cells depicting astrocytes ingroups of vehicle or indicated compound treated mice.

FIGS. 17A-17B show representative images of NF-kB p65+ microglia inmouse hippocampus. FIG. 17A shows representative IHC section stained forCD11b+ (red) and p65+(brown) microglia. FIG. 17B shows the mean opticaldensity of the 3, 3′-diaminobenzidine (DAB)-positive cells depicting p65microglia in groups of vehicle or indicated compound treated mice.

FIGS. 18A-18E show the effect of GILZ analogs (PGA 2 and PGA 4)treatment on cytokines in brain tissues in LPS inducedneuroinflammation: Adult C57Bl/6 mice were subjected to LPS inducedneuroinflammation and treated with the indicated GA or control peptidesas described in materials and methods. Protein extracts of brain tissuesharvested at the end of experiment was assessed for indicatedpro-inflammatory cytokines (IL-1β=FIG. 18A, IL-6=FIG. 18B, TNF-α=FIG.18C, and IFN-γ=FIG. 18D) and activated p65 NF-Kβ subunit (FIG. 18E).Values are average/−S.D. *=p<0.05 as compared to vehicle treated mice,@=p<0.05 as compared with CP-1 or CP-2 Or CP-3 treated mice.

FIGS. 19A-19E show the effects of GILZ analogs (PGA 2 and PGA 4)treatment on apoptosis related molecules in brain tissues of LPS inducedneuroinflammation. Groups of adult C57Bl/6 mice were subjected to LPSinduced neuroinflammation and treated with the indicated GA or controlpeptides as described in materials and methods. Equal quantity of cDNAisolated from brain tissues of mice treated with GA or CP were amplifiedfor IL-1β, CD14, Bclx1 and Bcl2 mRNA by quantitative PCR. The mRNAexpression in each sample was finally determined after correction withGAPDH expression. FIG. 19A shows gel electrophoresis of the PCR productsGAPDH (111 bp), IL-1β (89 bp), CD14 (206 bp), Bclx1 (84 bp) and Bcl2 (96bp). Relative mRNA quantitation of the indicated product with respect tothat of housekeeping gene GAPDH is shown for IL-1β (FIG. 19B), CD14(FIG. 19C), Bclx1 (84 FIG. 19D) and Bcl2 (FIG. 19E). Data areaverage±SD. *=p<0.05 with respect to vehicle and CP treated group.

FIGS. 20A-20D show DEX at 1 μM was not toxic but at higher (10 μM)concentration reduced the viability of SK-N-SH cells by 25% (FIG. 20A).The GILZ protein and the transcript was increased at lower but decreasedat the highest DEX concentration tested (FIG. 20B-20D).

FIGS. 21A-21H show GILZ properties observed in the cortex, hippocampusand cerebellum. (FIG. 21A and FIG. 21B): Immunohistochemitry of theadult C57BL/6 mouse brain shows positive staining for GILZ in the CA1,CA3 and DG regions in hippocampus; (FIG. 21C): Reduced GILZ inhippocampus following LPS induced neuroinflammation. (a,b,c: insets showhigh power) (FIG. 21D): Negative control. (FIG. 21E): Relative GILZ mRNAin the brain tissues of control and LPS induced mice (N=6). (FIG. 21F):Gel electrophoresis of the PCR products. Correlation of GILZ mRNA withCD14 mRNA is shown in FIG. 21 G and with CD4 mRNA is shown in FIG. 21H.

FIGS. 22A-22D show immunohistichemical staining. Immunohistichemicalstaining for GILZ (FIG. 22A & FIG. 22B) and for phosphorylated NF-κB p65(FIG. 22C & FIG. 22D) of brain from 5×FAD (FIG. 22B, FIG. 22D) mice andnon-transgenic liter mates (FIG. 22A, FIG. 22C). The expression of GILZwas lower and that of the p65 was higher in 5×FAD mice as compared tothat in non-transgenic liter mates. Insets a,b,c and d show magnifiedimages.

FIGS. 23A-23B show that inflammatory cytokines IFN-γ and IL-17 areelevated brain homogenates (FIG. 23A) and injured spinal cordhomogenates (FIG. 23B) harvested at day 7 post-SCI (see left bars ineach pair). GILZ analog was administered at 6 days post-SCI and reducedIFN-γ and IL-17 cytokine levels in brain tissue and spinal cord tissue(see right bars in each pair).

FIGS. 24A-24B show microglial response in the hippocampus (FIG. 24A) andlesion epicenter of the spinal cord (FIG. 24B) 7 days post-SCI.Microglia are labeled with Iba-1 antibody (red) and blue represents cellnuclei (10×).

The following numbered embodiments are contemplated for the presentdisclosure and are non-limiting:

1. A pharmaceutical composition comprising a polypeptide from about 8 toabout 12 amino acid residues, the polypeptide comprising a tetrapeptidehaving the sequence of PXXP, wherein

P is proline; and

X is any amino acid.

2. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of Formula I: X1-X2-X3-P-X4-X5-P-X6-X7, whereineach of X1, X2, X3, X4, X5, X6, and X7 is independently any amino acid.

3. The pharmaceutical composition of clause 2, wherein X1 is E.

4. The pharmaceutical composition of clause 2, wherein X1 is A.

5. The pharmaceutical composition of any of clauses 2 to 4, wherein X2is P.

6. The pharmaceutical composition of any of clauses 2 to 4, wherein X2is A.

7. The pharmaceutical composition of any of clauses 2 to 6, wherein X3is A.

8. The pharmaceutical composition of any of clauses 2 to 6, wherein X3is L.

9. The pharmaceutical composition of any of clauses 2 to 6, wherein X3is K.

10. The pharmaceutical composition of any of clauses 2 to 9, wherein X4is selected from the group consisting of R, L, E, A, and Y.

11. The pharmaceutical composition of any of clauses 2 to 10, wherein X5is selected from the group consisting of Q, A, and S.

12. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of XXXPXXPXX.

13. The pharmaceutical composition of clause 12, wherein the polypeptidecomprises a sequence of EPAPXXPXX (SEQ ID NO: 1).

14. The pharmaceutical composition of clause 12, wherein the polypeptidecomprises a sequence of EPLPXXPXX (SEQ ID NO: 2).

15. The pharmaceutical composition of clause 12, wherein the polypeptidecomprises a sequence of EAAPXXPXX (SEQ ID NO: 3).

16. The pharmaceutical composition of clause 12, wherein the polypeptidecomprises a sequence of APAPXXPXX (SEQ ID NO: 4).

17. The pharmaceutical composition of clause 12, wherein the polypeptidecomprises a sequence of APKPXXPXX (SEQ ID NO: 5).

18. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of Formula II: X1-X2-X3-P-X4-X5-P-X6-X7-X8, whereineach of X1, X2, X3, X4, X5, X6, X7, and X8 is independently any aminoacid.

19. The pharmaceutical composition of clause 18, wherein X1 is E.

20. The pharmaceutical composition of clause 18, wherein X1 is A.

21. The pharmaceutical composition of any one of clauses 18 to 20,wherein X2 is P.

22. The pharmaceutical composition of any one of clauses 18 to 20,wherein X2 is A.

23. The pharmaceutical composition of any one of clauses 18 to 22,wherein X3 is A.

24. The pharmaceutical composition of any one of clauses 18 to 22,wherein X3 is L.

25. The pharmaceutical composition of any one of clauses 18 to 22,wherein X3 is K.

26. The pharmaceutical composition of any one of clauses 18 to 25,wherein X4 is selected from the group consisting of R, L, E, A, and Y.

27. The pharmaceutical composition of any one of clauses 18 to 26,wherein X5 is selected from the group consisting of Q, A, and S.

28. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of XXXPXXPXXX.

29. The pharmaceutical composition of clause 28, wherein the polypeptidecomprises a sequence of EPAPXXPXXX (SEQ ID NO: 6).

30. The pharmaceutical composition of clause 28, wherein the polypeptidecomprises a sequence of EPLPXXPXXX (SEQ ID NO: 7).

31. The pharmaceutical composition of clause 28, wherein the polypeptidecomprises a sequence of EAAPXXPXXX (SEQ ID NO: 8).

32. The pharmaceutical composition of clause 28, wherein the polypeptidecomprises a sequence of APAPXXPXXX (SEQ ID NO: 9).

33. The pharmaceutical composition of clause 28, wherein the polypeptidecomprises a sequence of APKPXXPXXX (SEQ ID NO: 10).

34. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPRQPAT (SEQ ID NO: 11).

35. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPRAPEG (SEQ ID NO: 12).

36. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPLAPYG (SEQ ID NO: 13).

37. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPRAPGT (SEQ ID NO: 14).

38. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPRAPDG (SEQ ID NO: 15).

39. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPLPEAPDT (SEQ ID NO: 16).

40. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPESPQV (SEQ ID NO: 17).

41. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPEQPDG (SEQ ID NO: 18).

42. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of APAPASPQV (SEQ ID NO: 19).

43. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EAAAESPQV (SEQ ID NO: 20).

44. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of APAPAAPET (SEQ ID NO: 21).

45. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EAAAEAAET (SEQ ID NO: 22).

46. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPEAPEGY (SEQ ID NO: 23).

47. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPYQPEG (SEQ ID NO: 24).

48. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAYEAQET (SEQ ID NO: 25).

49. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPEAGET (SEQ ID NO: 26).

50. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPEAPET (SEQ ID NO: 27).

51. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPESPQV (SEQ ID NO: 17).

52. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of EPAPYQPRG (SEQ ID NO: 28).

53. The pharmaceutical composition of clause 1, wherein the polypeptidecomprises a sequence of APKPYQPRG (SEQ ID NO: 29).

54. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPRQPAT (SEQ ID NO: 11).

55. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPRAPEG (SEQ ID NO: 12).

56. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPLAPYG (SEQ ID NO: 13).

57. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPRAPGT (SEQ ID NO: 14).

58. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPRAPDG (SEQ ID NO: 15).

59. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPLPEAPDT (SEQ ID NO: 16).

60. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPESPQV (SEQ ID NO: 17).

61. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPEQPDG (SEQ ID NO: 18).

62. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of APAPASPQV (SEQ ID NO: 19).

63. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EAAAESPQV (SEQ ID NO: 20).

64. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of APAPAAPET (SEQ ID NO: 21).

65. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EAAAEAAET (SEQ ID NO: 22).

66. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPEAPEGY (SEQ ID NO: 23).

67. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPYQPEG (SEQ ID NO: 24).

68. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAYEAQET (SEQ ID NO: 25).

69. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPEAGET (SEQ ID NO: 26).

70. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPEAPET (SEQ ID NO: 27).

71. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPESPQV (SEQ ID NO: 17).

72. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of EPAPYQPRG (SEQ ID NO: 28).

73. The pharmaceutical composition of clause 1, wherein the polypeptideconsists essentially of a sequence of APKPYQPRG (SEQ ID NO: 29).

74. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPRQPAT (SEQ ID NO: 11).

75. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPRAPEG (SEQ ID NO: 12).

76. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPLAPYG (SEQ ID NO: 13).

77. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPRAPGT (SEQ ID NO: 14).

78. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPRAPDG (SEQ ID NO: 15).

79. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPLPEAPDT (SEQ ID NO: 16).

80. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPESPQV (SEQ ID NO: 17).

81. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPEQPDG (SEQ ID NO: 18).

82. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of APAPASPQV (SEQ ID NO: 19).

83. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EAAAESPQV (SEQ ID NO: 20).

84. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of APAPAAPET (SEQ ID NO: 21).

85. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EAAAEAAET (SEQ ID NO: 22).

86. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPEAPEGY (SEQ ID NO: 23).

87. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPYQPEG (SEQ ID NO: 24).

88. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAYEAQET (SEQ ID NO: 25).

89. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPEAGET (SEQ ID NO: 26).

90. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPEAPET (SEQ ID NO: 27).

91. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPESPQV (SEQ ID NO: 17).

92. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of EPAPYQPRG (SEQ ID NO: 28).

93. The pharmaceutical composition of clause 1, wherein the polypeptideconsists of a sequence of APKPYQPRG (SEQ ID NO: 29).

94. The pharmaceutical composition of any one of clauses 1 to 93,wherein the composition suppresses p65 binding.

95. The pharmaceutical composition of any one of clauses 1 to 93,wherein the composition suppresses p65 activation.

96. The pharmaceutical composition of any one of clauses 1 to 93,wherein the composition inhibits NF-κB translocation to the nucleus of acell.

97. A pharmaceutical formulation comprising the pharmaceuticalcomposition of any one of clauses 1 to 96.

98. The pharmaceutical formulation of clause 97 further comprising apharmaceutically acceptable carrier.

99. The pharmaceutical formulation of clause 97 or clause 98 optionallyincluding one or more other therapeutic ingredients.

100. The pharmaceutical formulation of any one of clauses 97 to 99wherein the formulation is a single unit dose.

101. A lyophilisate or powder of the pharmaceutical formulation of anyone of clauses 97 to 100.

102. An aqueous solution produced by dissolving the lyophilisate orpowder of clause 101 in water.

103. Use of the pharmaceutical composition or the pharmaceuticalformulation of any one of clauses 1-100 for the treatment of aneurodegenerative disease in a patient.

104. The use of clause 103 wherein the neurodegenerative disease isAlzheimer's Disease.

105. The use of clause 103 wherein the neurodegenerative disease isParkinson's Disease.

106. The use of clause 103 wherein the neurodegenerative disease ismultiple sclerosis.

107. The use of clause 103 wherein the neurodegenerative disease isamyotrophic lateral sclerosis (ALS).

108. A method of treating a neurodegenerative disease in a patient, saidmethod comprising the step of administering the pharmaceuticalcomposition or the pharmaceutical formulation of any one of clauses1-100 to the patient in need thereof.

109. The method of clause 108 wherein the neurodegenerative disease isAlzheimer's Disease.

110. The method of clause 108 wherein the neurodegenerative disease isParkinson's Disease.

111. The method of clause 108 wherein the neurodegenerative disease ismultiple sclerosis.

112. The method of clause 108 wherein the neurodegenerative disease isamyotrophic lateral sclerosis (ALS).

113. Use of the pharmaceutical composition or the pharmaceuticalformulation of any one of clauses 1-100 for the treatment of a spinalcord injury in a patient.

114. The use of clause 113, wherein the administration of thepharmaceutical composition reduces inflammation in the spinal cord ofthe patient.

115. The use of clause 113, wherein the administration of thepharmaceutical composition reduces inflammation in the brain of thepatient.

116. The use of clause 113, wherein the administration of thepharmaceutical composition reduces the presence of one or more cytokinesin the spinal cord of the patient or in the brain of the patient.

117. The use of clause 116, wherein the cytokine is IFN-γ.

118. The use of clause 116, wherein the cytokine is IL-17.

119. A method of treating a spinal cord injury in a patient, said methodcomprising the step of administering the pharmaceutical composition orthe pharmaceutical formulation of any one of clauses 1-100 to thepatient in need thereof.

120. The method of clause 119, wherein the administration of thepharmaceutical composition reduces inflammation in the spinal cord ofthe patient.

121. The method of clause 119, wherein the administration of thepharmaceutical composition reduces inflammation in the brain of thepatient.

122. The method of clause 119, wherein the administration of thepharmaceutical composition reduces the presence of one or more cytokinesin the spinal cord of the patient or in the brain of the patient.

123. The method of clause 122, wherein the cytokine is IFN-γ.

124. The method of clause 122, wherein the cytokine is IL-17.

In particular, the following analogs were developed and evaluated:

Analog Sequence SEQ ID NO: Name Analog 1 EPAPRQPAT 11 Analog 2 EPAPRAPEG12 Analog 3 (GA-2) EPAPLAPYG 13 PGA-2 Analog 4 EPAPRAPGT 14 Analog 5EPAPRAPDG 15 Analog 6 EPLPEAPDT 16 Analog 7 (GA-4) EPAPESPQV 17 PGA-4Analog 8 (GA-5) EPAPEQPDG 18 PGA-5 Analog 9 APAPASPQV 19 Analog 10EAAAESPQV 20 Analog 11 APAPAAPET 21 Analog 12 EAAAEAAET 22 Analog 13EPAPEAPEGY 23 Analog 14 (GA-3) EPAPYQPEG 24 PGA-3 Analog 15 EPAYEAQET 25Analog 16 EPAPEAGET 26 Analog 17 EPAPEAPET 27 Analog 18 EPAPESPQV 17Analog 19 EPAPYQPRG 28 Analog 20 (GA-1) APKPYQPRG 29 PGA-1

Furthermore, the following analogs were tested:

Name Sequence SEQ ID NO: PGA-1 APKPYQPRG 29 PGA-2 EPAPLAPYG 13 PGA-3EPAPYQPEG 24 PGA-4 EPAPESPQV 17 PGA-5 EPAPEQPDG 18

Increased NF-κβ p65 secondary to aging and environmental stimulicontribute significantly to the inflammation and degeneration in AD.Synthetic compounds including terpenoids like adenanthin and resveratrolor other sirtuin activators interact with NF-κβ p65, suppressinflammation and cytotoxicity in AD models. As opposed to highthroughput screening, computational design of interface peptide mimicsfollowed by functional evaluation represents an efficient method in thedrug design and discovery process. Here we report the design andphysicochemical characteristics of peptide analogs of the GILZ:p65interface, show that select analogs bind p65-TAD and suppress Aβ inducedtoxicity in human fetal brain cells exhibiting potential therapeuticvalue for AD.

In human interactome, preponderant transient intermolecular interactionsare mediated by proline rich epitope of one protein binding the aromaticresidue rich flat interface of the second protein. Often the prolinerich epitope adopts an extended PP_(II) helical conformation thatbehaves as an adaptable glove in obtaining the correct bindingorientation. Here it is pertinent to note that the template basedmodeling suggested that the p65 binding domain of GILZ exhibits aPP_(II) helical conformation and that the p65-TAD is unstructured orflat. Mutational analyses identified ¹²⁰PEA(S)P¹²⁴ of GILZ as hot spotresidues for interacting with NF-κB p65. In the GILZ:p65-TAD complex,the critical proline of GILZ, ¹²⁰P exhibits φ and ψ angles of −67°±5° of142.5°±15° respectively and is in close proximity with the conservedphenylalanines (⁵³⁴F, ⁵⁴²F) of p65-TAD. Collectively, these observationssuggest that the GILZ:p65-TAD complex represents a druggable target fordevelopment of specific therapeutic leads.

Incorporating rational substitutions in the polyproline motif of GILZ inthe context of the p65-TAD interface we designed multiple peptideanalogs of GILZ or GA. Molecular superposition is one of the mostimportant means to interpret the relations between three-dimensionalstructures. The low RMSD upon superimposition with experimental PP_(II)and wild type GILZ suggests that the select GA represent true structuralmimic of the p65 binding domain of GILZ. Significantly docking analysesshowed that the top ranked GA exhibited >20% contact with thefunctionally critical p65-TAD residues (F⁵³⁴, F⁵⁴²) in >90% of dockedsolutions.

An important advantage of proline-rich motif at interface of transientintermolecular interactions is the weak binding kinetics withoutcompromising affinity. Furthermore, it allows for introduction of smallchanges in the sequence of the motif or its binding domain to mediatelarge changes in the affinity of the interaction. We observed that theGA-1 and GA-2 evaluated by functional analysis exhibited greateraffinity for binding r-p65 than the full length r-GILZ. Previously,proline rich peptides that bind Src homology 3 binding domain or thetranscription factor human estrogen receptor alpha or the cell surfaceCD80 ligand have been shown to exhibit dissociation constant (KD) in themicromolar range and inhibit protein:protein interactions.

Many peptide drugs including copaxone, leuprolide acetate/goserelin(peptide antagonists of GnRH receptor), octreotide (a cyclic octapeptidemimicking natural hormone somatostatin) and glucagon likepeptide-1(GLP-1) analog have been evaluated in models of ADsubstantiating the credibility of peptide drugs for AD. Proline richPP_(II) helical peptides such as apidaecin, oncocin and drosocin haveshown to exhibit significant influx into CNS and distribution within thebrain parenchyma. Hence PGA is likely to cross the blood brain barrierand reach optimal concentration in the brain to be clinically efficient.Significantly, the estimated LD50 for GA-1 and GA-2 as determined by themethod of Speilmann et al. is within the range of these peptide drugs.Furthermore the ability of GA-1 and GA-2 to suppress Aβ inducedinflammatory and cytotoxic responses in human fetal brain cells suggestspotential as AD therapeutic agents.

The goal of biological therapies is to restore healthy balance bytargeting specific molecules that are critical for mediating orperpetuating imbalanced cellular responses. In recent yearspeptide-based drugs have gained considerable value in the discoveryphase of drug development, in particular in the design of interfacemimotopes. The functionally active peptides are amenable to furthermodifications into peptidomimetic compounds or small molecules withimproved pharmacokinetic properties. In this context, the low molecularweight GA-1 and GA-2 can act as lead compounds in the development ofspecific small molecule inhibitors of NF-κβ p65 with significanttherapeutic potential for chronic neurodegenerative diseases includingAD (see FIG. 7).

In some embodiments, the pharmaceutical formulations described hereinfurther comprise a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical formulations described herein furthercomprise a pharmaceutically acceptable diluent. Diluent or carrieringredients used in the pharmaceutical compositions containingpolypeptides can be selected so that they do not diminish the desiredeffects of the polypeptide. Examples of suitable dosage forms includeaqueous solutions of the polypeptides, for example, a solution inisotonic saline, 5% glucose or other well-known pharmaceuticallyacceptable liquid carriers such as alcohols, glycols, esters and amides.

As used herein, “carrier” refers to any ingredient other than the activecomponent(s) in a formulation. Pharmaceutically acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition(see, e.g., Remington's Pharmaceutical Sciences, 17th ed. (1985)). Thechoice of carrier will to a large extent depend on factors such as theparticular mode of administration, the effect of the carrier onsolubility and stability, and the nature of the dosage form. In oneillustrative aspect, the carrier is a liquid carrier.

As used herein, the term “pharmaceutically acceptable” includes“veterinarily acceptable”, and thus includes both human and animalapplications independently. For example, a “patient” as referred toherein can be a human patient or a veterinary patient, such as adomesticated animal (e.g., a pet).

In some embodiments, the pharmaceutical formulations described hereinoptionally include one or more other therapeutic ingredients. As usedherein, the term “active ingredient” or “therapeutic ingredient” refersto a therapeutically active compound, as well as any prodrugs thereofand pharmaceutically acceptable salts, hydrates, and solvates of thecompound and the prodrugs. Other active ingredients may be combined withthe described polypeptides and may be either administered separately orin the same pharmaceutical formulation. The amount of other activeingredients to be given may be readily determined by one skilled in theart based upon therapy with described polypeptides.

In some embodiments, the pharmaceutical formulations described hereinare a single unit dose. As used herein, the term “unit dose” is adiscrete amount of the composition comprising a predetermined amount ofthe described polypeptides. The amount of the described polypeptides isgenerally equal to the dosage of the described polypeptides which wouldbe administered to an animal or a convenient fraction of such a dosagesuch as, for example, one-half or one-third of such a dosage.

In one illustrative aspect, parenteral formulations may be suitablyformulated as a sterile non-aqueous solution or as a dried form to beused in conjunction with a suitable vehicle such as sterile,pyrogen-free water. The preparation of parenteral formulations understerile conditions, for example, by lyophilization, may readily beaccomplished using standard pharmaceutical techniques well known tothose skilled in the art.

The aqueous preparations according to the invention can be used toproduce lyophilisates by conventional lyophilization or powders. Thepreparations according to the invention are obtained again by dissolvingthe lyophilisates in water or other aqueous solutions. The term“lyophilization,” also known as freeze-drying, is a commonly employedtechnique for presenting proteins which serves to remove water from theprotein preparation of interest. Lyophilization is a process by whichthe material to be dried is first frozen and then the ice or frozensolvent is removed by sublimation in a vacuum environment. An excipientmay be included in pre-lyophilized formulations to enhance stabilityduring the freeze-drying process and/or to improve stability of thelyophilized product upon storage. For example, see Pikal, M. Biopharm.3(9)26-30 (1990) and Arakawa et al., Pharm. Res., 8(3):285-291 (1991).

In one embodiment, the solubility of the polypeptides used in thepreparation of a parenteral formulation may be increased by the use ofappropriate formulation techniques, such as the incorporation ofsolubility-enhancing agents.

In various embodiments, formulations for parenteral administration maybe formulated to be for immediate and/or modified release. Modifiedrelease formulations include delayed, sustained, pulsed, controlled,targeted and programmed release formulations. Thus, a polypeptide may beformulated as a solid, semi-solid, or thixotropic liquid foradministration as an implanted depot providing modified release of theactive compound.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials. The formulations can also bepresented in syringes, such as prefilled syringes.

In various embodiments, the dosages of the polypeptides can varysignificantly depending on the patient condition and the severity of thedisease to be treated. The effective amount to be administered to apatient is based on body surface area, patient weight or mass, andphysician assessment of patient condition.

Suitable dosages of the polypeptides can be determined by standardmethods, for example by establishing dose-response curves in laboratoryanimal models or in humans in clinical trials. Illustratively, suitabledosages of polypeptides (administered in a single bolus or over time)include from about 1 pg/kg to about 10 μg/kg, from about 1 pg/kg toabout 1 μg/kg, from about 100 pg/kg to about 500 ng/kg, from about 1pg/kg to about 1 ng/kg, from about 1 pg/kg to about 500 pg/kg, fromabout 100 pg/kg to about 500 ng/kg, from about 100 pg/kg to about 100ng/kg, from about 1 ng/kg to about 10 mg/kg, from about 1 ng/kg to 1mg/kg, from about 1 ng/kg to about 1 μg/kg, from about 1 ng/kg to about500 ng/kg, from about 100 ng/kg to about 500 μg/kg, from about 100 ng/kgto about 100 μg/kg, from about 1 μg/kg to about 500 μg/kg, or from about1 μg/kg to about 100 μg/kg. In each of these embodiments, dose/kg refersto the dose per kilogram of a patient's or animal's mass or body weight.

The present disclosure provides uses and methods of using thepharmaceutical compositions, and uses and methods of usingpharmaceutical formulations comprising the pharmaceutical compositions,for the treatment of neurodegenerative diseases such as Alzheimer'sDisease, Parkinson's Disease, multiple sclerosis, and amyotrophiclateral sclerosis (ALS).

The present disclosure also provides uses and methods of using thepharmaceutical compositions, and uses and methods of usingpharmaceutical formulations comprising the pharmaceutical compositions,for treating a spinal cord injury in a patient. In some embodiments, theadministration of the pharmaceutical composition reduces inflammation inthe spinal cord of the patient. In some embodiments, administration ofthe pharmaceutical composition reduces inflammation in the brain of thepatient. In various embodiments, administration of the pharmaceuticalcomposition reduces the presence of one or more cytokines in the spinalcord of the patient or in the brain of the patient. In certainembodiments, the cytokine is IFN-γ. In other embodiments, the cytokineis IL-17.

EXAMPLES Example 1

Peptides and reagents: All GILZ peptides were synthesized as peptideamides with amino-terminal acetylation (Genescript, Piscataway, N.J.) at95% purity as confirmed by mass spectrometry. To facilitateintracellular delivery the GA were either co-synthesized with the cellpenetrating agent, TAT (transactivator of transcription) peptide or usedas covalent mixture with Pep-1 chariot peptide (Anaspec, Fremont,Calif.). Recombinant human p65 protein (r-p65) with DDK tag (catalognumber TP320780), purified recombinant human GILZ protein (r-GILZ) withGST tag and biotinylated anti-DDK antibody were from Ori-GeneTechnologies Inc., Rockville, Md. Purified Aβ₁₋₄₂ peptide was purchasedfrom American Peptide company (American peptide company, Sunnyvale,Calif.: Product #62-0-80 Lot #1310160T). Aβ₁₋₄₂ peptide stock (1 mg/mL)was prepared in cell culture medium and incubated at 37° C. for 24 hoursprior to use in cell cultures.

Comparative modeling: Models of human GILZ and its mimics were built bythe CPH models and Geno3D servers using delta sleep inducing peptide(DSIP-PDB:1DIP) as template based on >90% sequence similarity. While theGeno3D system builds models based on ‘topology mapping, the CPH systemuses profile-based alignment as seed for developing energy-minimizedhomology model. The secondary structure assignment of the GILZ modelswas independently assessed by the PROSS (Protein dihedral angle-basedSecondary Structure assignment) program. Superimposition of the model ofeach GILZ mimic with experimentally determined PP_(II) helix and wildtype GILZ determined the similarity between the structures in terms ofroot mean square deviation (RMSD). Homology models of p65-TAD wasdeveloped similarly using elongation factor eEF3 (PDB:3H7H) as templatewith which it shares 42% sequence similarity.

Design and modeling of GILZ mimic: Strategies to determine the smallestbiologically active fragment of a lead peptide involves truncation,deletion, alanine scanning and substitution of residues. Deletionstudies suggested that the amino terminal helix of GILZ is critical fordimerization but is not involved in the interaction with NF-κβ.Truncation mutants suggested that the residues spanning ¹²⁰P-P¹²⁷ of theGILZ were critical for GILZ mediated inhibition of NF-κβtransactivation. Molecular modeling showed that the GILZ-COOH or a 22residue peptide derived from the proline rich region adopted an extendedPP_(II) helical conformation and interacted with the p65-TAD exhibitingweak binding kinetics. Alanine scanning mutagenesis suggested that thesubstitution of the ¹²⁰PXXP¹²³ motif abrogated the ability to inhibitNF-κβ transactivation potentially due to loss of PP_(II) conformation.In human and mouse GILZ, hydrogen bonding between the side chain of Seror Thr and the backbone carbonyl of Glu or Pro respectively couldcontribute to the stability of PP_(II) conformation. We designed 40 GILZmimics by incorporating rational substitutions in the p65 binding motifof GILZ with residues that increase the propensity for PP_(II) helicalconformation and stabilize it. Comparative modeling with substitutedresidues for all 40 GA was performed to obtain structural representationof each with reference to the adjacent residues of human GILZ. Inaddition we introduced conformational constraints by superimposing eachGILZ mimic model on structures with solved or experimental PP_(II) helixand wild type human GILZ to select for mimics with significantstructural homology (see FIGS. 1A-1F). The PP_(II) content of GILZmimics as determined by PROSS ranged from 14.3%, 28.6% and 42.9%. SincePP_(II) helix formation is a locally driven event with little/noinvolvement of long-range interactions, it is logical to presume thatthe synthetic GILZ mimetic peptide with blocked end groups will adopt asimilar conformation as in the predicted model. Twenty GILZ mimics thatexhibit near structural congruence with DSIP (<1 Å) or wild type GILZ orexperimentally determined PP_(II) structure (<2 Å) were selected for insilico docking.

Example 2

GILZ:p65-TAD docking: Models of human GILZ or GILZ mimic and the p65-TADwere applied as probe and target respectively in PatchDock, a geometrybased algorithm that yields docked transformations scored on the basisof molecular shape complementarity and atomic desolvation energy. Topone thousand solutions were refined using FireDock (Fast InteractionRefinement in molecular docking), a program that optimizes binding ofthe probe by restricting side-chain flexibility to clashing interfaceresidues. The refined docking solutions were scored based on softenedvan der Waals interactions, atomic contact energy, electrostatic andadditional binding free energy estimations. The top ranked solutions soobtained were further screened using Chimera for interatomic distance of<5 Å between the residues of GILZ mimic and the functionally criticalresidues of p65-TAD. The solution with most contacts was further refinedby FlexPepDock using the wild type GILZ:p65-TAD complex with greaterthan 50% intermolecular residue contacts as reference.

Docking of GILZ mimic and p65-TAD: To be of potential therapeutic value,the GILZ mimic should adopt PP_(II) helical conformation in the contextof the critical binding residues in p65-TAD. The p65-TAD is commonlydivided into two distinct regions, TAD-1521-551 consisting of 36 aminoacids and TAD-2428-520 with 92 residues. It has been reported that theTAD₁ accounts for nearly 95% of the transactivation potential offull-length p65 and that the TAD2 alone is less potent mediating about30% activation. In particular, the highly conserved aromatic residues(F⁵³⁴, F⁵⁴²), acidic residues (D⁵³¹,D⁵⁵³) and phosphorylation sites(Ser⁵²⁹,Ser⁵³⁶) in p65-TAD1 have been identified as critical fortransactivation.

Homology model of p65-TAD was built using solution structure of theelongation factor eEF3 (PDB: 2XI3, 2WI3) with which it shares 42%sequence similarity. The spatial orientations of wild type GILZ and top20 GILZ mimics with p65-TAD were assessed by multiple dockingalgorithms. One thousand interaction possibilities identified byrigid-docking algorithm were improved by coarse refinement to restrictside-chain flexibility at the interface. The docked complexes wereranked using an optimized global energy function for higher probabilityprediction. Top ten solutions of each GILZ mimic were evaluated forproximity to p65-TAD residues. In general two residues are considered incontact with each other if the distance between the CP atoms is <5 Å.Interactions between the conserved F⁵³⁴-F⁵⁴² in the p65-TADi and thecritical prolines P¹²⁰/P¹²³ of GILZ could promote C-H⋅⋅π interaction andprovide substantial binding energy in the GILZ:p65-TAD complex. Allsolutions that exhibited an RMSD of <5 Å with the critical F⁵³⁴ and F⁵⁴²were selected for further screening (Table 1).

TABLE 1 Characteristics of GILZ mimics p65-Transactivation DomainSuperimposition % TA2 GILZ % TA1 CR-1 CR-2 CR-3 % PPII PPII model521-531 435-455 462-479 491-505 PGA-1 14.3 0.82 0.216 19 43 39 20 PGA-242.9 0.531 0.216 18 32 13 9 PGA-3 14.3 0.109 0.428 11 14 23 4 PGA-4 14.31.049 0.482 4 13 47 29 PGA-5 28.6 1.234 0.586 11 14 23 4 PGA-6 14.30.844 0.645 21 54 34 9 PGA-7 14.3 0.156 0.681 30 20 0 11 PGA-8 14.3 1.720.842 15 41 32 5 PGA-9 42.9 0.257 0.88 24 42 29 13 PGA-10 28.6 0.161 0.912 38 23 7 PGA-11 28.6 0.703 0.912 20 46 23 6 PGA-12 42.9 1.17 1 10 1218 6 PGA-13 14.3 0.58 1.018 3 35 55 6 PGA-14 28.6 1.172 1.033 32 28 10 2PGA-15 14.3 0.391 1.047 12 36 8 7 PGA-16 14.3 0.252 1.154 18 38 33 1PGA-17 28.6 1.293 1.227 15 25 24 9 PGA-18 14.3 1.248 1.243 16 24 31 22PGA-19 28.6 0.581 1.49 7 22 24 7 PGA-20 42.9 1.174 1.613 37 43 9 2

Table 1 shows the characteristics of twenty GILZ mimics: GILZ models(GM) with substituted residues at the proline rich region of wild typeGILZ sequence were developed using Geno3D and CPHModels. Each GA modelwas superimposed over wild type GILZ and experimentally determinedpolyproline (PP_(II)) helical structures. The root mean square deviation(RMSD) of each superimposition as a measure of structural similarity isshown. Each GM was docked with the molecular model of p65-TAD and thedocked complexes were screened for interface p65-TAD residues within 5 Ådistance of GM.

Sixty of the top one hundred predictions of wild type GILZ exhibitedclose proximity with nearly 50% of p65-TADi residues suggestingnear-native interactions (FIG. 2A). The wild type GILZ:p65-TAD complexwith the lowest global energy and maximum contacts with p65-TAD wasselected as reference for refining each of the 20 GILZ mimic-p65TADcomplexes in two hundred independent FlexPepDock simulations.Significantly ten of the twenty GILZ mimics exhibited interatomicdistance of <5 Å not only with the conserved phenylalanine in p65-TADibut also with the putative LXXLL motif in p65-TAD2 (FIGS. 2B and 2C).The LXXLL motif commonly observed in transcription factors are known tomediate protein-protein interactions. A rank order was developed basedon percent PP_(II) content, structural similarity with wild type GILZand percent contact with p65-TAD. We selected two GILZ mimics or analogs(GA-1 and GA-2) that exhibit near native docking, structural congruencewith experimental PP_(II) and wild type GILZ and good PP_(II) potentialfor screening by cellular analyses. In addition we selected one mimicwith good PP_(II) potential but fewer contacts with p65-TAD and two GILZmimics that possess low PP_(II) content but acceptable percent contactwith the p65-TAD in docking analyses as control peptides for PP_(II)conformation (control peptide 1) and p65 docking potential (controlpeptide 2 and control peptide 3) respectively.

Example 3

Binding of GILZ and human r-p65: Previously we observed that the r-GILZexhibits ten times higher affinity than a 22 residue GILZ peptide forr-p65. We used similar method to determine direct binding kinetics of GAhexapeptides with human r-p65. High binding ELISA plates were coatedwith increasing concentrations of r-GILZ (0.5 μM to 20 μM) or each GA orcontrol peptide (20 μM-640 μM) and probed with 80 pM r-p65 followed bydetection with anti-DDK antibody. Absorbance at 650 nm was measured witha mixing time of 30 s using BIORAD microplate reader. Percent bound p65was determined considering the binding response of r-GILZ (20 pM) withr-p65 as 100%. The dissociation constant of the interaction between theGA or the control peptide and r-p65 was determined by the method ofFriguet et al., as described. A fraction of the bound r-p65 (x) and theratio of bound r-p65 to the free GA or control peptide (y) wasdetermined by the equations: x=(A₀−A)/(A₀), where A₀ is the absorbanceof r-p65anti-p65 complex in the absence of bound GA or control peptideand y=(A₀−A)/(a₀−i₀)×(A₀−A)/(A₀), where a₀ is the total concentration ofGA or control peptide and i₀ is the total concentration of r-p65. KD forthe interaction was determined by the Scatchard equation: x=1+K_(D)/y.

Kinetics of GA: p65 interaction: Previously the strength of interactionbetween rGILZ or wild type GILZ-P and r-p65 has been shown to be5.91±2.4*10⁻⁷M and 1.12±0.25*10⁻⁶M, respectively. We evaluated thebinding kinetics of individual GA at increasing concentrations with theplate bound r-p65 protein at constant concentration. We observed thatthe percent bound r-p65 was over 25% with GA-1, GA-2 and CP-1 even atthe lowest concentration evaluated (FIG. 3A). The dissociate constant,KD, as calculated by the method of Friguet et al. was 2.29+/−0.2×10⁻⁶Mfor GA-1 and 3.24+/−0.19×10⁻⁶M for GA-2. The strength of interaction ofthe GA:p65-TAD binding is consistent with other transientprotein:peptide interactions such as that of peptide inhibitors ofmatrix metalloproteinase and that of SMRT peptides binding the BTBdomain of BCL6. The control peptides exhibited higher K_(D) values of3.27+/−1.8×10⁻⁶M, 3.4+/−0.15×10⁻⁶M and 4.28+/−0.5×10⁻⁶M for CP-1, CP-2and CP-3 respectively (FIGS. 3B-3F).

Example 4

Cell Titer-Glo (CTG) luminescent cell viability assay: Humanneuroblastoma (SK-N-SH) cells cultured in minimal essential medium (MEM)supplemented with 1% fetal bovine serum (FBS) and 1% penicillin (100U/ml)/streptomycin (100 μg/ml) were differentiated with 10 μM all-transretinoic acid for 7 days. After resting for 24 hours in the low serummedium, the cells were seeded in 24 well (10⁵ cells/well) culture platesin fresh medium and incubated with 50 μM or 500 μM of individual GA orcontrol peptide for additional 24 hours. The cultures were thenphotographed using a phase contrast Leica microscope (Leica MicrosystemsInc, Buffalogrove, Ill.). Subsequently, the cells were harvested, lysedwith lysis buffer (M-PER, Pierce) and the lysate was assessed formetabolic activity using the luciferase based CTG assay (CTG, Promega,Madison, Wis.). Briefly, cell lysate (5 μL) in 25 μL of phosphatebuffered saline was transferred to an opaque white 96-well plate, andthen 30 μL of CTG assay solution was added. The relative luminescentsignal (RLT) was quantified using a Glowmax luminometer (Promega).

Effect of GA on cellular morphology and metabolic activity: Any compoundwith potential therapeutic effect should be biocompatible and nontoxic.So, we first screened the effects of GA on cellular morphology andviability. Cultures of neuroblasts exposed to 50 μM or 500 μM ofindividual GA or CP-1 or CP-2 did not show any change in cell morphologysuggesting that the four peptides were not toxic. Exposure to CP-3 ateither concentration showed morphological changes consistent with celldeath (FIG. 4A). The effect of GA on cellular metabolic activity wasassessed by the CTG assay which measures intracellular ATPconcentrations as an indicator of actual cell number. The results of theCTG assay were very similar and showed that the two GA and CP-1 and CP-2did not adversely affect the differentiated neuroblastoma cells, buttreatment with CP-3 reduced the viability of the cells at bothconcentrations tested (FIG. 4B).

Example 5

Lactate dehydrogenase (LDH) assay: To detect direct GA-induced celllysis we performed LDH release assays (Roche Molecular Diagnostics).Human glioblastoma (U373) cells were maintained in MEM supplemented with10% FBS and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml) at 37° C.in 5% CO₂-humidified incubators and sub-cultured once or twice a week.Approximately 5×10⁴ U373 cells/well were cultured in 96-well plates inthe presence of increasing concentrations of individual GA or controlpeptide from 0.5 μM to 500 μM. Cells treated with 2% triton-X 100 (SigmaAldrich, St. Louis, Mo.) for 10 minutes served as positive control.Untreated cells served as controls for spontaneous LDH-release. SpecificLDH-release was calculated according to the following formula:LDH-release %=100*(GA or control peptide treated cells—untreatedcells)/(positive control-untreated cells). The IC50 values wereextrapolated by logarithmic estimation. The LD50 in mg/kg was predictedusing the formula, Log (LD50)=0.506×(log IC50)+0.475.

Detection apoptosis by flow cytometry: To further assess cellcytotoxicity, the apoptotic effects of individual GA or control peptidewas evaluated by the Annexin-V and propidium iodide (PI) dual stainingmethod (Annexin-V-Fluos staining kit, Roche Diagnostics, Mannheim,Germany). As opposed to apoptotic cells, necrotic cells with rupturedcell membrane take up PI, the DNA binding dye. Thus, cells which take upboth fluorochromes are a mixture of apoptotic and necrotic cells,whereas cells that exclude PI but bind Annexin V are (early) apoptoticcells. U373 cells cultured with varying concentrations of individual GAor control peptide (0.5 μM to 50 μM) for 24 hours were centrifuged andsuspended in 100 μl of Annexin V/PI labelling solution (20 μl each ofAnnexin-V-Fluos labelling reagent and PI in 1 ml of binding buffer) for15 min at room temperature. After washing the cells were resuspended inPBS:1% paraformaldehyde and analyzed using a FACScan flow cytometer (BDBiosciences, San Jose, Calif.).

Effect of GA on membrane integrity and apoptosis: Membrane integrity asa measure of cell survival was assessed by the leakage of intracellularLDH molecules into culture medium. Treatment with GA-1 and GA-2 werebest tolerated as indicated by the reduced % LDH release at allconcentrations as compared to untreated cultures or cultures exposed tocontrol peptides (FIG. 5A). IC50 extrapolated from regression analysisis 226.78 μM for GA-1 and 198.4 μM for GA-2 (FIGS. 5C and 5D). The IC50was lower for CP-1 (49.9 μM), CP-2 (26.58 μM) and CP-3 (26.63 μM) (FIG.5E-5G). Using the Speilmann method, the LD50 in mg/kg body weight isestimated to be 380.44 for GA-1 and 369.42 for GA-2 and 272.78, 237.52and 237.62 for CP-1, CP-2 and CP-3, respectively. Many therapeuticpeptides such as leuprolide and glatiramer acetate have been shown toexhibit similar LD50 values. Furthermore the cytotoxic effect of eachpeptide at increasing concentration from 0.5 μM to 50 μM was assessed byAnnexin and PI staining (FIG. 5B). GA-1 and GA-2 exhibit <25% apoptosisat highest concentration.

Example 6

Functional assays in human primary mixed brain cultures (HFB). Primarycultures of mixed HFB were prepared from the brain parenchyma of abortedfetuses (80-100 days gestational age). The tissues were obtained fromthe Birth Defects Research Laboratory (BDRL) at the University ofWashington, Seattle with approval from the Indiana UniversityInstitutional Review Board (IRB). The IRB determined that the use ofanonymous human biological materials received from this depository isnot considered as human subjects research (supplementary document, s1).The NIH funded BDRL tissue distribution program operates separately infull compliance with all relevant state and federal laws and regulationswith donors providing written informed consent.

Fetal brain materials (10-20 g) were shipped overnight in chilledHibernate-E medium (Invitrogen) supplemented with B27 (Invitrogen),GlutaMAX (Invitrogen) and antibiotic-antimycotic solution (Cellgro). HFBwere prepared and cultured as described. Briefly, cells were cultured inNeurobasal medium (Invitrogen) without phenol red supplemented with1×B27, 50 mM GlutaMAX, 1×antibiotic cocktail, 5 ng/mL recombinantfibroblast growth factor 2 (bFGF) (Invitrogen), and 2 μL/mL Normocin(InVivoGen, San Diego, Calif., USA). Cells were counted and seeded ontopoly-D-lysine (Sigma-Aldrich) coated 24-well plates (Corning, Lowell,Mass., USA) at 1.5×10⁵ cells per well and maintained at 37° C. in a 5%CO₂ incubator. Half media changes were performed every 4th day ofculture and morphology was monitored via phase contrast microscopy.Culture medium was removed from cells on day 17 (DIV17) and replacedwith Neurobasal medium with B27. Appropriate wells were then addedvehicle, or carrier peptide or individual GA or control peptide (at 50μM) for 30 min followed by exposure to Aβ₁₋₄₂ at a final concentrationof 10 μM/well and incubated for 4 hours or 48 hours. Cells andconditioned media were harvested and stored for further analysis.Relative ATP concentration was measured using the CTG kit (Promega).Data is presented as ΔRLU=RLU of Aβ₁₋₄₂ exposed cells-RLU unexposedcells. Conditioned media collected were assessed for specific cytokinesusing the OptEIA kits (BD Biosciences). Nuclear and cytoplasmicfractions were extracted using the CelLytic™ NuCLEAR™ Extraction Kit(Sigma) following manufacturer's protocol. Five microgram of nuclearextracts was incubated in a 96-well plate coated with oligonucleotidescontaining the NF-κβ consensus nucleotide sequence (5′-GGGACTTTCC-3′)(SEQ ID NO: 30). The activated NF-κβ bound to DNA was detected byanti-p65 antibody followed by a peroxidase coupled secondary antibodyand substrate using the TransAM kit protocol (Active Motif). Nuclearextracts of Raji cells was used as the positive control.

Select GA protect against Aβ₁₋₄₂ induced toxicity in human fetal braincells. We used an in-vitro neurodegeneration model in which primaryhuman fetal brain cells are allowed to mature gradually. The systemprovides an opportunity to test the pharmacological and toxicologicaleffects of GA on differentiated neurons and glia simultaneously. Therelative ATP concentration of GA or control peptide treated HFB culturesin the presence or absence of Aβ₁₋₄₂ over the cultures exposed tovehicle alone (A-RLU) was determined by the CTG assay. Cultures exposedto GA were best tolerated while the cultures treated with CP-2 or CP-3exhibited significant toxicity (FIG. 6A). The mean RLU of vehicletreated cells varied between 7352.75+/−1265.2 and 16157.5+/−4950 and theaverage RLU of cultures exposed to Aβ₁₋₄₂ varied between6067.25+/−903.05 and 11574.25+/−4139.3 in different experiments. Theviability was significantly higher in cells exposed to Aβ₁₋₄₂ andtreated with GA-1 or GA-2 (FIG. 6B). Although A-RLU was higher incultures treated with CP-1, it was not significant when compared to thatin untreated Aβ₁₋₄₂ exposed cultures. The relative concentration ofIL-10 and IL-6 was significantly lower in culture medium of cellsexposed to Aβ₁₋₄₂ and treated with GA-1 or GA-2 as compared with thatfrom untreated or cultures treated with the control peptides (FIGS. 6Cand 6D).

Select GA treatment inhibits activated p65: Previously Aβ₁₋₄₂ has beenshown to enhance expression of activated NF-κB in glia and post-mitoticneurons. We measured nuclear p65 binding activity using activated NF-κBspecific ELISA. Nuclear p65 was significantly higher in cells exposed toAβ₁₋₄₂ than in unexposed cells (FIG. 6E). There was a trend towardsdecreased nuclear p65 in Aβ₁₋₄₂ exposed cells treated with GA-1 and GA-2as compared to untreated cells or cells treated with control peptide. Nosignificant difference in nuclear p65 was observed in cells treated onlywith the peptides in the absence of exposure to Aβ1-42.

Comparative analysis of physical and functional characteristics of knownreceptor antagonists and peptide drugs in clinical use today with thatof GA, suggest that the GA-1 and GA-2 exhibit significant drug likeproperties (Table 2).

TABLE 2 Physical and functional characteristics of GILZ analog andcontrol peptides Structural similarity RMSD Wild % type PPII Dockingfeatures # rotatable K_(D) LD50 apoptosis GILZ peptide % TA1 % TA2 %PPII bonds Log P (uM) (mM) at LD50 Overall <1 >1 <1 >1 >25 <25 >25<25 >25 <25 <10 >10 <5 >5 <3 >3 >100 <100 <25 >25 rank 1 0 1 0 1 0 1 0 10 1 0 PGA1 1 1 1 0 0 0 0 0 0 0 3 PGA2 1 1 1 1 1 1 1 1 1 1 10 PGA3 1 0 10 1 0 1 0 1 1 6 PGA4 1 1 1 1 1 1 1 0 1 1 9 PGA5 1 1 0 1 0 0 0 0 1 0 4

Example 7

Design and selection of PGA: Human GILZ has an amino terminal a-helixfor dimerizing and a carboxy terminal proline glutamic acid rich regionfor intermolecular interactions. Mutational and binding analyses showedthat the proline rich region of GILZ adopts an extended PP_(II) helicalconformation and binds the p65-TAD. In the GILZ:p65 complex, interactionbetween the imino ring of P¹²⁰/P¹²³ of GILZ and the side chain ofF⁵³⁴/F⁵⁴² in the p65-TAD could promote C-H⋅π interactions and providesubstantial binding energy. Functional studies have shown that thehighly conserved F⁵³⁴ and F⁵⁴² in the transactivation domain arecritical for p65 induced transactivation.

Forty PGA were designed by incorporating rational substitutions in thep65 binding domain of GILZ with residues that facilitate PP_(II)formation and stabilization at the interface with the p65-TAD. Homologymodels of PGA built using delta sleep inducing peptide (DSIP; PDB:1DIP)as template were screened for conformational constraints bysuperimposing on experimental PP_(II) and wild type human GILZ. ThePP_(II) content of all PGA as determined by PROSS method ranged from14.3%, 28.6% and 42.9%. Since PP_(II) helix formation is often a locallydriven event, the synthetic PGA with blocked end groups will likelyadopt a similar conformation as in the predicted model. Twenty PGA thatexhibit structural congruence with DSIP (<1 A), wild type GILZ andexperimental PP_(II) structure (<2 Å) were selected for dockinganalysis.

Homology model of p65-TAD was built using elongation factor eEF3(PDB:3H7H) as template. Spatial orientation of wild type GILZ peptide oreach PGA with p65-TAD was assessed by multiple algorithms includingrigid-docking based on shape complementarity and coarse refinementmethods that restrict side-chain flexibility at interface. The dockedcomplexes were ranked using an optimized global energy function.Significantly most solutions of top ten PGA exhibited close proximity tothe phenylalanines (F⁵³⁴, F⁵⁴²) and the putative LXXLL motif in p65-TAD(see FIGS. 9C and 9D). The LXXLL motif in transcription factors has beenshown to mediate protein-protein interactions. A rank order developedbased on percent PP_(II) content, structural similarity with wild typeGILZ and percent contact with p65-TAD identified five PGA with nearnative docking and good PP_(II) potential. The top five PGA wereselected for cellular analyses (see FIGS. 8A-8D).

Example 8

Kinetics of PGA:-p65 interaction: The binding kinetics of top five PGAwith full length human r-p65 was assessed as described. Plate boundGILZ-P or PGA at increasing concentrations was probed with r-p65-DDK(Ori-Gene Technologies Inc., Rockville, Md.) at constant concentrationand detected with anti-DDK. The percent bound r-p65 increased withincreasing concentration of PGA (see FIG. 9A). The dissociationconstant, KD, calculated by the method of Friguet, was lowest for PGA-2(2.29+/−0.2×10⁻⁶M) followed by PGA-4 (3.28+/−0.2×10⁻⁶M), PGA-5(3.4+/−0.2×10⁻⁶M), PGA-3 (3.27+/−1.8×10⁻⁶M) and PGA-1 (4.28+/−0.5×10⁻⁶M)(FIGS. 9B-9C).

Example 9

Effects of PGA on membrane integrity and apoptosis: The top five PGAwere covalently synthesized with the cell penetrating TAT(transcriptional transactivation) peptide to facilitate intracellulardelivery (Genescript, N.J.). Membrane integrity of U373 microglial cellsexposed to increasing concentration of each PGA from 0.5 μm to 500 μMwas assessed by the leakage of intracellular lactate dehydrogenase (LDH)into culture medium (Roche, Ind.).

All five PGA exhibited a dose dependent LDH release (FIG. 10A). IC₅₀extrapolated from regression analysis was highest for PGA-1 (2.27 μM)followed by PGA-5 (2.0 μM), PGA-2 (1.95 μM), PGA4 (1.5 μM) and PGA-3(0.98 μM) (FIG. 10B). The cytotoxic effect of each PGA at increasingconcentration from 0.5 μM to 50 μM was also assessed by Annexin and PIstaining. PGA-2 and PGA-4 exhibit <25% apoptosis.

Example 10

Select PGA protects against Aβ₁₋₄₂ induced toxicity, prevents p65activation and cytokine response in human fetal brain (HFB) cells.Primary HFB cells pretreated with vehicle or each PGA at IC₅₀concentration were cultured in the presence of Aβ₁₋₄₂ aggregates (10 μM)for 24 hours at 37° C. and 5% CO₂. Viability in terms of relativeluminescent Units (RLU) was quantified using a Glowmax luminometer(Promega, Wis.). Data are presented as ΔRLU=RLU of Aβ₁₋₄₂ exposedcells-RLU of unexposed cells. HFB cultures exposed to PGA-2, PGA-3 andPGA-4 were well tolerated. Cultures treated with PGA-1 and PGA-5exhibited significant toxicity (FIG. 11A). The mean RLU of vehicletreated HFB varied between 7352.75+/−1265.2 and 16157.5+/−4950. AverageRLU of cultures exposed to Aβ₁₋₄₂ varied between 6067.25+/−903.05 and11574.25+/−4139.3 in different experiments. The viability wassignificantly higher in HFB cells treated with PGA-2, PGA-3 or PGA-4 andexposed to Aβ₁₋₄₂ (FIG. 11B). The culture supernatants were assessed forcytokines by ELISA (R&D Systems, Minn.). AIL-1p (not shown) and AIL-6concentration was significantly lower in PGA-2, PGA-4 or PGA-5 treatedcultures over unstimulated or cultures exposed to Aβ₁₋₄₂ alone (FIG.11C). Primary HFB cultured similarly and harvested at the end of 4 hourswas assessed for nuclear p65 using TransAM kit for NF-κB p65 (ActiveMotif, Calif.). Aβ₁₋₄₂ exposed cells treated with PGA-2 and PGA-4exhibited decreased nuclear p65 (FIG. 11D).

Example 11

Evaluation of the effect of PGA-2 and PGA-4 on AD-relevant cells. Anin-vitro neurodegeneration model derived from primary HFNC can beutilized in the instant example. Previously maturing HFNC cultures havebeen shown to exhibit positive staining for neuronal, glial and synapticmarkers. Further, the neuronal population has been shown to increasewith time and exhibit physiological activity. In addition the cells arecapable of being transfected with external agents providing a tool forevaluating the efficacy of potential drug candidates for CNSpathologies. Importantly, abundant Aβ production and tau proteins makethis a powerful model for Alzheimer's research. The effects of PGA-2 andPGA-4 on neuronal and glial cell morphology, BACE-1 and Aβ production inprimary HFNC can be evaluated. (see FIGS. 13A-13E).

HFNC: Briefly, human brain cells derived from aborted fetus (90-110 daysgestational age) can be plated in poly D-lysine coated 24-well plates(Sigma, St. Louis, Mo.) using defined culture medium (Neurobasal mediumwith 1×B27, 50 mM Glutamax, 1×antibiotics cocktail and recombinant humanbasic fibroblast growth factor 2 (bFGF) at 5 ng/mL (Gibco, Grand Island,N.Y.) and incubated at 37° C., 5% CO₂. Half media changes can beperformed every fourth day and morphology monitored by phase contrastmicroscopy. The procedure usually results in ˜75% pure neurons. Purityof the cultured cells has been demonstrated by visualization of neuronsand glia using pan-neuronal antibody (Millipore, Billerica, Mass.) andglial fibrillary acidic protein (GFAP) (Sigma) respectively.

PGA treatment and immunocytochemistry: PGAs were covalently synthesizedwith the cell penetrating TA T to facilitate intracellular delivery aspeptide amides with amino terminal acetylation. At DIV16 (mixedpopulation of neuronal and glial cells) or DIV20 (predominantly matureneurons) of HFNC, the defined culture medium can be replaced withNeurobasal medium supplemented only with B27. PGA-2 (168.9 μM), PGA-4(101.7 μM) at IC50 concentration or TAT peptide (100 μM) can be addedand the cultures continued to be incubated for 24 hours. The effect onneuronal and glial cell morphology can be assessed by immunostainingusing fluorophore conjugated astroglial markers (GFAP) and neuronspecific proteins (Nestin, Pan N). Stained sections can be visualizedwith an inverted fluorescent microscope (Leica Microsystems) andappropriate sets of filters.

Quantitation of BACE-1 and AB: Separate HFNC cultures at Div16 can beadded PGA-2, PGA-4 or TAT (carrier). Equal quantity of cDNA isolatedfrom cells harvested after 24 hours can be amplified for BACE-1, APP andreference genes β-2 microglobulin and GAPDH by real time polymerasechain reaction (Applied Biosystems; assay IDs: human BACE-1(Hs00201573_m1), human APP (Hs01552283_m1), human GAPDH (4333764T),human B2M (4333766T). Fold changes can be calculated using the delta-Ctmethod normalized to the geometric mean of the reference genes. Levelsof Aβ₁₋₄₀ and Aβ₁₋₄₂ in the culture medium can be measured usingspecific commercially available ELISA kits (Covance, Princeton, N.J.).Absolute Aβ values (in pg/ml of culture medium) can be normalized tototal protein and scaled relative to levels in untreated cultures.

As PGA-2 or PGA-4 did not affect the morphology of HFNC as seen underinverted bright field microscope, HFNC exposed to either PGA may exhibitsignificant neuronal staining on day-20 suggesting neuropreservation.Transfection of human brain cells with NF-κB dependent miRNA has beenshown to downregulate BACE-1, APP and AP production. By blocking p65,PGA-2 and PGA-2 treatment may reduce BACE-1 transcription and consequentAβ production.

Example 12

Investigation of the molecular effects of PGA on AD relevant cells:Accumulating environmental and oxidative stress induces NF-κB p65activation in neuronal and glial cells contributing to AD pathogenesis.Quantitation of biologically active molecules integrally involved in ADpathology is used as outcome measures in early stages of drug discovery.

Cell culture: HFNC cultures at Div16 added PGA-2 or PGA-4 at IC50 or TAT(carrier) as above can be exposed 30 minutes later to H₂O₂ (10 μM) orsodium nitroprusside (SNP) (30 pM) as radical oxygen or nitric oxidedonor respectively for 4 hours or 24 hours. Some cultures can be added acell permeable 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (10 μM)(Molecular Probes, Eugene, Oreg.) probe at the time of PGA treatment tomeasure reactive oxygen species (ROS). Cells and culture medium can becollected for RNA and protein analysis.

Evaluation of activated NF-κB. cytokines and glutamate: The effect ofPGA on p65 transactivation in each cell type can be evaluated byco-labeling of cells harvested at 4 hours using fluorophore conjugatedmarkers for neuron (NSE, Nestin, Pan N), astrocytes (GFAP); microglia(IBA1) and nuclear p65 (Abcam Cambridge, Mass.) and visualized with aninverted fluorescent microscope. Activated p65 in nuclear fraction ofcells can be quantitated by ELISA. IFN-α, IL-iβ and IL-6 and glutamatein culture medium collected at 24 hours can be measured using specificELISA kits (R&D Systems, Minn.) and enzymatic assays (BioVision Inc.Milpitas, Calif.) respectively.

Cytoprotection assays: Fluorescence in cell lysates added DCFH-DA andcollected at 4 hours can be measured at 504/529 nm and data expressed asratio of ROS to total protein. Activated caspase in culture medium canbe measured using caspase-3 assay kit (R&D systems). Total proteinisolated from cells harvested at 24 hours can be probed by immunoblot(Abcam) for apoptosis related proteins Bax, Bcl-2 and caspase-3 usingspecific antibodies. Fold changes in these apoptotic proteinstranscripts can be determined by polymerase chain reaction arrays(Applied Biosystems; Human TaqMan® Human Apoptosis Array; Cat.#4414072).

Other neuroprotective agents suppressed caspase-3 and Bax in neuronalcells. Preliminary data suggest that PGA-2 and PGA-4 may suppresscytokines and cytotoxic mediators. Alternatively primary rat corticalneuronal cells (PRCN) can be used to evaluate the effects of PGA.

Example 13

Evaluation of the In-Vivo Efficacy of PGA in a Transgenic Mouse Model ofAD.

Selection of Animal Model: The R1.40 APP YAC transgenic mice created byintroducing entire genomic copies of mutant human APP (with K670N/M671LFAD substitutions) into the mouse genome can be utilized in the instantexample. As compared to the cDNA based hAPP transgenic mice, the R1.40APP YAC transgenic mice exhibit regional AO distribution, preferentialdeposition of Aβ₁₋₄₂, overlapping deposition of APOE, extensive neuriticabnormalities, and increased markers of inflammation mimicking manycharacteristics of human AD. Inhibition of NF-κB by tolfenamic acid hasbeen shown to reduce cerebral Aβ plaque burden in homozygous R1.40 mice.

Experimental procedures:

Animals: Starter pair of R1.40 [B6.129-Tg(APPSw)40Btla/Mmjax mice (Stock#034831)] can be purchased from Jackson Laboratory (Bar Harbor, Me.).Breeding colonies can be genotyped and maintained under standard animalhousing conditions in a 12-hour dark light cycle with free access tofood and water at the laboratory animal resource facility (LARC) at IUSchool of Medicine (IUSM). The background strain for R1.40 is mixed,which can serve as non-AD controls. All experiments can be conductedaccording to the Institutional Animal Care and Use Committee (IACUC) andapproved by the local ethics committee.

PGA dosing and treatment regimen: The LD50 calculated using the in-vitroIC50 values and the regression equation [log (LD50)=0.506×log(IC50)+0.47551] for PGA-2 and PGA-4 equals 1396.83 mg/kg and 1693.87mg/kg bodyweight respectively. Appropriate dose of PGA-2 and PGA-4 canbe dissolved in 100 μl of sterile PBS for i.p administration. R1.40 APPYAC transgenic mice develop parenchymal and vascular amyloid deposits by12-13 months and exhibit reactive gliosis by 14-16 months. Four groupswith 7 homozygous mutant APP YAC transgenic mice each can beadministered daily either saline, PGA-2, PGA-4 or carrier peptide (TAT)at 1300 mg/kg (equivalent to LD50 of PGA-2) for four weeks beginning atweek 40.

Histology: Mice can be perfused transcardially with ice cold PBS 4%paraformaldehyde at the end of four weeks. Brain tissues can beharvested, frozen and stored for histological analysis. Sections ofentorhinal cortex, orbitofrontal cortex, and frontal cortex can bestained for congophilic dense-core amyloid plaques and immunostained forAβ plaques and inflammatory markers including Iba1, IL-6, IL-1β, TNF-α,and NF-κB-P65.

Photomicrographs can be analyzed for percent stained area for plaqueload and inflammation. An inflammatory focus is defined as the presenceof clusters of 20 or more aggregated mononuclear cells. The total numberof inflammatory foci can be counted from 3 different sections of eachspecimen.

Leuprolide (5 g/kg) and glatiramer acetate (230 mg/kg) administered i.p.for similar periods were efficacious in models of AD. These peptideshave been measured in the cerebrospinal fluid in optimum concentrationssuggesting efficient CNS delivery and substantiating the credibility ofpeptide drugs for AD. Based on the mechanisms of actions of GILZ andpreliminary data, percent stained area of congo red positive plaques, Aβplaques and inflammation may be significantly reduced in PGA treatedmice.

Example 14

Transformation of PGA into small molecule agents: PGA peptides arerationally designed structural mimics of GILZ in the contest of thep65-TAD binding interface. Specific inhibition of activated p65 by PGAhas been shown to suppress inflammatory and cytotoxic responses in-vitroand in-vivo.

Three dimensional structure databases incorporated within the modelingprograms of CAVEAT and PHASE can be searched to identify templates withbonds that adopt PP_(II) orientation and serve as attachment points. Thefunctional groups of critical proline of human GILZ or the substitutedPGA residue can be grafted onto the non-peptide scaffold. The resultantPGA pharmacophore can be characterized by binding affinity andfunctional studies. A similar approach has been previously usedsuccessfully in developing inhibitors of Factor Xa for treatingcoagulation pathologies.

PepMMsMIMIC is a virtual screening platform tool used to identifypharmacophore or chemical compounds that mimic a natural peptide orprotein in 3D space. Using this tool, a three dimensional similaritysearch of PGA-2/PGA-4 structure can be initiated amongst seventeenmillion conformers calculated from commercially available chemicals. Toppeptide mimetic can be ranked by two different scoring functions takinginto account high electrostatic, chemical and shape complementaritytogether with pharmacophore fingerprints similarity. Using this method,multiple small molecule nutlin analogues have been identified aspeptidomimetics of MDM2/p53 interaction. Preliminary search suggeststhat the chemical moiety, methyl-N-butyl-N[2,6-di-nitro-4-(trifluormethyl)phenyl] carbamate, may be similar toPGA-2 peptide.

Modified amino acids or incorporation of enantiomers may be used toenhance identification of PGA specific pharmacophore.

Example 15 Suppression of Pathology in an Alzheimer's Disease Model

Peptides: GA-1 and GA-2 are peptides designed to mimic the PP_(II)helical orientation of the p65 binding motif of the GILZ. The controlpeptides include sequence control consisting of residues with littlepropensity for PP_(II) helix (CP-1) formation and/or for binding p65-TAD(CP-2) or unstructured peptide with no p65 binding potential (CP-3). Allpeptides were co-synthesized with the cell penetrating agent TAT(GRKKRRQRRRPQ, SEQ ID NO: 31) for intracellular delivery as peptideamides with amino terminal acetylation (Genescript, Piscataway, N.J.).All peptides were purified by semipreparative RP-HPLC and the identityof the purified peptide was confirmed by mass spectrometry.

Animals and lipopolysaccharide induced neuroinflammation: Male C57/6Jmice weighing 32 to 37 grams were housed five per cage at a temperatureof 22±2° C. with 12 hour light/12 hour dark cycles with free access tolaboratory food and water. Lipopolysaccharides (LPS) from Escherichiacoli 026:B6, =10,000 EU/mg, purified by phenol extraction was purchasedfrom Sigma (Sigma Aldrich, Mo., USA). The mice were randomly dividedinto seven groups: LPS, LPS+GA-1 or GA-2 or CP-1 or CP-2 or CP-3 or TAT.The LPS was administered intraperitoneally at 250 mg/kg in 100 μl ofsterile saline followed by corn oil in the morning daily for six days.The GA or CP was administered intravenously at 25 mg/kg in 100 μl ofsterile saline on alternate days. Additional control group included micereceiving saline alone. All animals were sacrificed on day 7.

Histology and immunohistochemistry: The mice were sacrificed on day 7 byintra-cardial perfusion of 25 ml of 0.9% saline. Brain from each mousewas removed, one half was immediately frozen and the other half fixed in4% paraformaldehyde for 24 h and later in 10% neutral buffered formalinfor additional 24 h. Fixed tissues were subsequently processed forparaffin embedding. Tissue sections (50 stained with Harris'shaematoxylin and counterstained with eosin were assessed forinflammation.

Immunohistochemistry: Serial 5μ thick coronal sections of each brainspecimen were immunostained for markers of microglial cells, astrocytesand NF-κB p65. Briefly, after deparaffinization and hydration sectionswere subjected to antigen retrieval by microwave incubation in 10 mMsodium citrate buffer (pH 6.0) followed by sequential incubation in 30%hydrogen peroxide and blocking buffer (Enzo biosciences,) to reducenon-specific binding. The sections were then stained with the followingprimary monoclonal antibodies from EMD Millipore Corporation, Temecula,Calif., USA: anti-Iba-1 (AIF1 clone: Cat. #MABN92) or anti-glialfibrillary acidic protein (GFAP) clone GA5 (Cat. #MAB3402, Millipore) oranti-CD11b (Cat. #MABF513) clone M1/70 or anti-NF-κB p65 subunit clone12H11 (Cat. #MAB3026). For mono-staining of Iba-1 and GFAP, detectionwas with the use of IHC select immunoperoxidase secondary detectionsystem (Millipore). The peroxidase conjugated streptavidin-biotin methodwas used and brown staining was considered positive. For dual stainingof CD11b and NF-κB p65 detection was achieved using the MULTIVIEW®(mouse-HRP/rabbit-AP) IHC kit (Catalog #: ADI-950-100, ENZOLifesciences, Farmingdale, N.Y., USA). For each marker, to confirm thespecificity of the antibodies, a separate set of sections from eachgroup were incubated with only the secondary antibodies, a condition inwhich no staining was present.

Quantitative analysis of staining intensity: Images of the IHC stainedsections were captured using the NIKON Multiphoton microscope withattached DS Ri2 camera. Five areas encompassing the hippocampus of eachbrain section were analyzed using the ImageJ Software. The number andthe relative optical density of Iba1+ and GFAP+ glial cells wasdetermined using the multi-point tool and the IHC-tool box. A trainingfor brown/red color was performed prior to measurement to define thethreshold for positive selection. Regions of interest encompassing thehippocampus were identified in each section, and the correspondingimages were quantified using integrated optical density, normalized tothe background optical density. Measurement of pixel intensity wasrestricted to the DAB or AP stained pixels of cells within definedcircularity. Five consecutive coronal sections were assessed for eachmouse.

Enzyme linked immunosorbent assay for cytokines: Frozen brain tissue washomogenized in RIPA lysis buffer (50 mmol/LTris-HCl, pH 6.8, 150 mmol/LNaCl, 5 mmol/L EDTA, 0.5% sodium deoxycholate, 0.5% NP-40) supplementedwith a cocktail containing protease and phosphatase inhibitors(Chemicon, Millipore) on ice and then centrifuged at 16,000×g for 30min. Supernatant was stored at −80° C. until further analysis. For eachsample, 10 μL of extracted protein was used for measuring the cytokinesIL-6, TNF-α, IFN-γ, IL-10 and TGF-β using specific OptEIA kits (BDBiosciences, Calif.).

NF-κB assay: Brain homogenates processed as above were assessed by theCell Signaling Technology PathScan® Phospho-NF-κB p65 (Ser⁵³⁶) SandwichELISA kit following manufacturer's protocol (Cell Signaling Technology,Boston, Mass.). Briefly, 40 μl of the extracted protein mixed with 60 μlof the sample diluent was added to phosphor p65 (Ser⁵³⁶) mouse mAbcoated microwells and incubated overnight at 4° C. After extensivewashing, the captured phospho-NF-κB p65 protein was detected byincubating with horse radish peroxidase (HRP) conjugated NF-κB p65rabbit mAb followed by washing and color development using the TMBsubstrate. Absorbance at 450 nM was measured using a microplate reader(Model 680; Bio-Rad Laboratories, Hercules, Calif., USA). Nuclearextracts of Raji cells was used as the positive control.

Real time polymerase chain reaction. Total cellular RNA was isolatedfrom each brain tissue using Qiagen kit (Invitrogen, Carlsbad, Calif.)following manufacturer's protocol and reverse transcribed using iScriptcDNA synthesis kit (Biorad, Calif.). Equal amount of cDNA measured bythe Nanodrop (ThermoFisher Scientific, Waltham, USA) was used foramplification of IL-1β, IL-12, CD14, Bcl_(XL) and Bcl2. Real time PCRwas performed using SYBR green/ROX qPCR master mix (SA Biosciences,Frederick, Md.) on the ABI Prism 7000 sequence detection system (AppliedBiosystem, Foster City, Calif., USA). Each reaction contains 2×12.5 μlof master mix, 1 μl of 10 μM of primers and 50 ng of the cDNA, to atotal volume of 25 μl. The thermal cycling conditions included aninitial denaturation step at 50° C. for 2 min, 95° C. for 3 min, 39cycles at 95° C. for 30 s, annealing temperature at 62° C. for 30 s andextension at 72° C. for 30 s.

The primers are F-βActin: (SEQ ID NO: 32)5′TCATGAAGTGTGACGTTGACATCCGTA3′; R-βActin: (SEQ ID NO: 33)5′CCTAGAAGCATTTG CGCTGCACGAT GG3′(102 bp); F-IL-1β: (SEQ ID NO: 34)5′AGCTGATGGCCCTAAACAGA3′; R-IL-1β: (SEQ ID NO: 35)5′GGTCGGAGATTC GTAGCTGG3′ (89 bp); F-CD14: (SEQ ID NO: 36)5′GAGCTAGACGAGGAAAGTTGT3′; R-CD14: (SEQ ID NO: 37)5′ACCGTAAGCCGCTTTAA GGACAGA3′ (206 bp); F-Bcl_(XL): (SEQ ID NO: 38)5′TGGAGTAAACTGGGGTCGCATC-3′; R-Bcl_(XL): (SEQ ID NO: 39)5′AGCCACAGTCATGCC CGTCAGG3′ (84 bp); and F-Bcl2: (SEQ ID NO: 40)5′CTCGTCGCTACCGTCGTCACTTCG3′; R-Bcl2: (SEQ ID NO: 41)5′GTGGCCCAGGTATG ACCCAG3′ (96 bp).

The gene specific threshold cycle (Ct) for each sample (ΔCt) wascorrected by subtracting the Ct for the housekeeping gene β-actin.Untreated controls were chosen as the reference samples. The ΔCt for allexperimental samples was subtracted by the ΔCt for the control samples(ΔΔCt).The difference in each gene specific threshold between the samplefrom vehicle treated and GA or CP treated cells was determined to obtainthe relative change in the specific mRNA. The magnitude of change in themRNA was expressed as 2^(−ΔΔ)Ct. Each measurement of a sample wasperformed in duplicates.

Example 16 Identification of Druggable Site: The Interface of p65:GILZInteraction

Structurally p65 has dimerizing amino terminal rel homology domain, anuclear localization sequence masked by the IκB inhibitory complex andcarboxy terminal TAD. The transactivation activity of p65 is mediated byinteractions of the TAD with co-regulators and the basal transcriptionmachinery. The p65-TAD is commonly divided into two distinct regions,TAD-1⁵²¹⁻⁵⁵¹ consisting of 36 amino acids and TAD-2⁴²⁸⁻⁵²⁰ with 92residues. While TAD1 accounts for nearly 95% of the transactivationpotential of full-length p65, TAD2 alone is less potent mediating about30% activation. In particular, the conserved aromatic residues (F⁵³⁴,F⁵⁴²), acidic residues (D⁵³¹, D⁵³³) and phosphorylation sites(Ser⁵²⁹,Ser⁵³⁶) in p65-TAD1 have been identified as critical fortransactivation.

GILZ is a p65-TAD binding protein that sequesters activated p65 andinhibits transactivation of inflammatory and apoptotic factors.Mutational, binding and structural studies suggested that the PER regionof GILZ adopts a PP_(II) helical conformation in the context of criticalphenylalanine in p65-TAD. PP_(II) helices highly represented atinterfaces of transient protein interactions in human proteome and oftenbehave as adaptable gloves in obtaining the correct binding orientation.The specificity of PP_(II) interaction is determined by residues of theinteracting partner. PP_(II) interface mimics constitute excellent drugtemplates. GA are rationally designed peptides with residuesubstitutions in the GILZ-PER with increased propensity to formstability of PP_(II) helical conformation in the context of p65-TAD (seeFIGS. 14A-14D). Screening 40 GA, we selected two analogs referred to asGA-1 and GA-2 that exhibit good PP_(II) potential, near native dockingwith p65-TAD, acceptable binding strength for transient interactions andinhibitory potential in cellular assays for evaluating the in-vivotherapeutic efficacy. In-silico predictions suggest that the predominantproteolytic products of GA-1 or GA-2 are to the right of thecell-penetrating carrier suggesting that the GA is potentially deliveredintracellularly as intact cargo to bind the activated p65 in thecytosol.

Example 17 GA Prevented LPS Induced Microglial and Astrocyte Activationin the Hippocampus

Peripherally-injected LPS induces a variety of central effects. Multipleinjections of LPS increase the number of F4/80+, CD11b+, or Iba1−+ cellsand induce morphological changes characteristic of activated microgliain the CNS. It was observed that the mice induced neuroinflammation andtreated with GA-1 or GA-2 exhibited reduced Iba1+ microglial cells andGFAP+ astroglial cells in the hippocampus as compared to that in thehippocampus of mice treated with control peptides or left untreated (seeFIGS. 15A-15B and FIGS. 16A-16B). Furthermore, it was observed that thenumber of CD11b+p65+ glial cells was significantly reduced in number andintensity in the hippocampus of GA-1 or GA-2 treated mice as compared tothat in untreated or control peptide treated mice (see FIGS. 17A-17B).

Example 18 GA Suppresses LPS-Induced Accumulation of InflammatoryProteins in the Hippocampus

Brain tissues were investigated for cytokines known to be critical formicroglial activation and gliosis in LPS-induced neuroinflammation.Brain tissue homogenates of GA-1 or GA-2 treated mice exhibited reducedIL-6 (18.7+/−2 pg/ml and 19.6+/−0.84 pg/ml respectively) treated mice ascompared to the homogenates of untreated (32.4+/−4.7 pg/ml) or controlpeptide treated mice (FIG. 18A). Similarly, brain homogenates of GA-1 orGA-2 treated mice exhibited reduced TNF-α (1500.1+/−476 pg/ml and1199.2+/−356.4 pg/ml respectively) as compared to the untreated(2287.7+/−473 pg/ml) or the control peptide treated mice (FIG. 18B). Theconcentration of IFN-γ was also significantly lower in brain tissue ofGA-1 (2863.2+/−697.5 pg/ml) or GA-2 (3125.1+/−815 pg/ml) treated mice ascompared to the untreated (4788.2+/−2069.1 pg/ml) mice (FIG. 18C). Theconcentration of IL-12 was also significantly lower in brain homogenatesof GA-1 or GA-2 treated mice (92.0+/−9.3 pg/ml, 101.3+/−15.9 pg/ml) ascompared with that in the brain tissues of untreated mice (132.9+/−17pg/ml) or CP treated mice (FIG. 18D).

Example 19 GA Treatment Suppressed Activated NF-κB p65 in LPS-InducedNeuroinflammation

Since LPS is a potent stimulator of NF-κB and GA are designed tosequester activated p65, the activated p65 in the brain homogenates wasmeasured using specific ELISA kits. It was observed that the absorbancesuggestive of activated p65 was significantly lower in the brain proteinextract from mice treated with GA-1 (0.098+/−0.03) or GA-2 (0.12+/−0.07)as compared to that of extract from mice left untreated (0.29+/−0.08) ortreated with CP-3 (0.23+/0.09) (FIG. 18E).

Example 20 GA Treatment Inhibited LPS-Induced Suppression ofAnti-Apoptotic Factors

To investigate the effects of GA on neurotoxicity, the expressions ofanti-apoptotic factors Bcl_(XL) and Bcl2 were determined by real timePCR. In addition, inhibition of LPS-induced cytokine secretioncorrelated with the decreased levels of steady-state mRNA for IL-1β inthe brain tissues of in GA-1 or GA-2 treated mice (FIGS. 19A & 19B). Itwas observed that the relative expression of CD14 mRNA was significantlylower in brain tissues of GA-1(0.48+/−0.1) or GA-2 (0.64+1-0.4) treatedmice as compared to that in the brain tissues of untreated (4.0+/−1.7)or control peptide (CP-1:1+/−0.3; CP-2:2.4+/−2; CP-3:1.2+/−0.1) treatedmice (FIGS. 19A & 19C). Relative expression of Bcl_(XL) and BCl2 wassignificantly higher in brain tissues of GA-1 (0.32+/−0.15 and1.25+/−0.5 respectively) or GA-2 (0.25+/−0.4 and 1.9+/−0.9 respectively)treated mice as compared to that in brain tissues of untreated(0.11+/0.1 and 0.22+/−0.5 respectively) or control peptide(CP-1:0.06+/−0.08 and 0.36+/−0.8, CP-2: 0.13+1-0.12 and 0.45+/−0.9;CP-3: 0.13+/−0.1 and 0.44+/−0.9 respectively) treated mice (FIGS. 19A,19D, & 19E).

Example 21 Investigation of the Role of GILZ in the GC Mediated NeuronalSurvival and Apoptosis

GILZ exhibits opposing effects of increased proliferation and reducedsurvival in many cell types. While GILZ induces apoptosis in thymocytes,it protected activated T cells from apoptosis. Although GILZ expressionin mouse brain has been observed, little is known about the role of GILZin neurons.

Preliminary results: The expression of GILZ in human neuroblastoma cellswas evaluated. SK-N-SH cells were cultured in minimal essential mediumand differentiated with 10 μM all-trans retinoic acid for 7 days asdescribed. After resting, the cells were cultured with increasingconcentrations of dexamethasone (Dex: 0-10 μM) for 24 h and viabilitywas determined by the MTT assay by measuring the absorbance of theintracellular formazon resulting from the reduction of the yellowtetrazolium MTT by the cell metabolic activity. Absorbance of vehicletreated cells was taken as 100% viable. GILZ expression was measured inprotein extracts from separate SK-N-SH cultures by Western blot analysisusing GILZ mAb [Santa Cruz Biotechnology, Dallas, Tex.]. Total RNAisolated from similar SK-N-SH cultures was reverse transcribed and theGILZ mRNA was amplified using the SYBR green qPCR mix (SA Biosciences,Fredrick, Md.) on the ABI Prism 7000 system (Applied Biosystem, Calif.).

The primers were: GILZ-F: (SEQ ID NO: 42) 5′-CATGGAGGTGGCGGTCTA TC-3′;GILZ-R: (SEQ ID NO: 43) 5′-CACCTCCTCTCTCACAGCGT-3′ and GAPDH-F:(SEQ ID NO: 44) 5′AGGTCGGTGTGAACGGATTT G-3′ and GAPDH-R: (SEQ ID NO: 45)5′-TGTAGACCATGTAGTTGAGGTCA-3′.Results: DEX at 1 μM was not toxic but at higher (10 μM) concentrationreduced the viability of SK-N-SH cells by 25% (FIG. 20A). The GILZprotein and the transcript was increased at lower but decreased at thehighest DEX concentration tested (see FIG. 20B-20D).

At low GC concentration, MR induced GILZ can facilitate neuronalsurvival, but in high GC state GILZ expression can be decreased withloss of protection.

The GILZ gene has six GRE in its promoter region and hence itsexpression is highly regulated by both endogenous and exogenous GC.Furthermore, complete concordance observed between the GILZ protein andthe mRNA implies that the regulation occurs at the transcriptionallevel. However, the dynamics of GILZ induction appear to vary indifferent tissues, suggesting that the local milieu could modulates itstranscription. The goal of this example is to characterize the functionsof neuronal GILZ and its regulation by GC.

Experimental Approach:

Culture of primary rat cortical neurons (PRCN): PRCN cultures can begenerated as described. Briefly, brain tissues dissected from a 16-weekold adult female Sprague-Dawley rat can be digested with papain.Neuronal cells isolated by gradient centrifugation using differentOptiprep concentrations can be cultured for 12 days in Neurobasal mediumsupplemented with bFGF and Normocin (InvivoGen, San Diego, Calif.) at37° C. and 5% CO₂ at which time point the cultures are rich in matureneurons. The purity of neurons can be identified by morphology (phasecontrast appearance as bright cells, with smooth rounded somata anddistinct processes) and immunocytochemical staining for NueN.Corticosterone (CORT) treatment: After transferring to serum freemedium, the effects of a range of CORT concentrations (1 nM-10 μM) oncellular viability, cytotoxicity and GILZ induction in PRCN cultures canbe analyzed. Since MR exhibits ten-fold higher affinity than GR for GC,we can next determine the relative contribution of MR and GR in GILZinduction. Separate PRCN cultures will be treated with MR antagonistspinorolactone (1 μM) or GR antagonist RU 486 (50 nM) prior to treatmentwith varying CORT concentration (1 nM-1 μM) to selectively block MR andGR respectively. In the preparation of CORT, RU486 and spinorolacotonethe concentration of 100% ethanol can be at <0.1% in the culture medium.

The PRCN cultures in each experiment can be harvested at the end of 24h, 48 h and 72 h. The primary read outs for these experiments caninclude i) assessment of cell viability and toxicity by MTT and lactatedehydrogenase (LDH) assays using cell lysates, ii) measurement of GRtransactivation in nuclear extracts by the enzyme-linked immunosorbentassay-based TransAM GR kit (Active Motif, Calif.), iii) quantitate GILZprotein by immunocytochemistry and immunoblot in cell lysates and iv)quantitate GILZ mRNA by RT-PCR.

The role of GILZ in the bimodal effects of glucocorticoid mediatedneuronal survival and apoptosis will be analyzed via overexpression ofGILZ in PRCN: PRCN can be transfected with pCMV6-GILZ-Myc-DDK usingTsc22d3 (NM 001077364) Mouse Tagged ORF Clone (CAT#: MG225004, OriGeneTechnologies Inc., Rockwille, Md.) as per manufacturer's protocol.Initial transduction at different multiplicity of infection can beperformed to determine the optimal transduction efficiency and GILZexpression. After resting, transfected cells can be exposed to 1 μM CORTfor 24 h. Equal amount of cDNA isolated from control and GILZoverexpressing cells can be used for gene expression studies byPathway-specific PCR arrays (RT² Neurotrophins & Receptors andApoptosis) to identify changes in genes related to survival and death ofneurons (Qiagen Inc, Germantown, Md.).

Cortisol treatment has been shown to mediate dose-dependent, bimodaleffects on neuronal cell viability and proliferation. Specifically, theprotective responses at low GC concentration is attributed to MRmediated transactivation. Pertinent to this study, in mouse hippocampalHT-22 cells that only express GR, GILZ was upregulated only in cellsstably transfected with MR. Hence, the GILZ mRNA can be upregulated inPRCN exposed to low dose CORT via MR. In lymphocytes, much like GC, GILZregulated apoptosis by modulating the ratio of anti-apoptotic (Bcl₂,Bcl_(XL)) to pro-apoptotic (Bax) molecules. Hence, the GILZoverexpression can suppress pro-apoptotic genes even in PRCN exposed tohigh dose of corticosterone.

Example 22 Pro-Inflammatory Role for Elevated GC in the CNS

Although traditionally conceptualized as anti-inflammatory agents,considerable evidence support a potent pro-inflammatory role forelevated GC in the CNS. This has been partially attributed to thereduced GR mediated transactivation of anti-inflammatory genes such asGILZ. GILZ overexpression or supplement suppressed inflammatorycytokines and pathology in models of arthritis and colitis.

Adult C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) wereadministered LPS (Sigma Aldrich, Mo.) intraperitoneally at 250 mg/kg for6 days. Serial 5μ thick coronal sections of brain from naïve and LPStreated mice were immunostained for GILZ using TSC22D3 mAb (Catalog #:H00001831, Abnova, Walnut, Calif.). The peroxidase conjugatedstreptavidin biotin method was used for detection and brown staining wasconsidered positive. Staining specificity was confirmed by incubationwith secondary antibody alone. Total RNA isolated from brain homogenateswas reverse transcribed. Equal amount of cDNA was used for amplificationof GILZ, CD14, CD4 and β-actin using SYBR green/Rox qPCR mix (SABiosciences) on the ABI 7000 system (Applied Biosystem).

Primers used were: F-βActin: (SEQ ID NO: 32)5′TCATGAAGTGTGA CGTTGACATCCGTA3′ R-βActin: (SEQ ID NO: 46)5′CCTAGAAGCATT TGCGCTGCACGATGG3′ (102 bp); F-CD14: (SEQ ID NO: 47)5′GATGACATGGTGA AGACGGC3′; R-CD14 (SEQ ID NO: 48)5′AGGCACAGGTCATCATCAA3′ (89 bp); F-CD4: (SEQ ID NO: 49)5′GAGAGTCAGCGGAGTTCT C3′; R-CD4: (SEQ ID NO: 50)5′CTCACAGGTCAAAGTATTGTTG3′ (92 bp); and F-GILZ: (SEQ ID NO: 51)5′AAGGCTAGCTCCGCAGGTGC-3′; R-GILZ: (SEQ ID NO: 52)5′AGGTGAGCGGCA CTCGGTCT3′ (120 bp).Fold change was determined by the 2^(−Δ)Ct method.

Results are shown in FIGS. 21A-21H. GILZ protein was observed in thecortex, hippocampus and cerebellum (FIG. 21A & 21B). LPS inducedneuroinflammation decreased GILZ expression (FIG. 21C). The GILZ mRNA inthe brain homogenates correlated inversely with that of CD4 and CD14,suggesting an inverse relationship between GILZ and inflammatorymarkers. FIG. 21D shows the negative control. FIG. 21E shows therelative GILZ mRNA in the brain tissues of control and LPS induced mice(N=6). FIG. 21F shows gel electrophoresis of the PCR products.Correlation of GILZ mRNA with CD14 mRNA is shown in FIG. 21G and withCD4 mRNA is shown in FIG. 21H.

GILZ Overexpression can Protect Against Neuronal Apoptosis:

Generation of conditional GILZ-transgenic (GILZ-Tg) mice: The standardCre/loxP systems can be used to develop conditional GILZ-Tg mice. Usinga Gateway-compatible ROSA26 locus targeting vector, transgenic mice canbe generated overexpressing GILZ in neurons. The target vector caninclude mouse Tsc22d3/Gilz-1 cDNA preceded by a loxP flanked stopcassette under control of the ROSA26 promoter followed by an IRES-EGFPcDNA. Embryonic stem cell clones with gilz transgene can be identifiedby Southern blotting. Conditional GILZ-Tg mice can be generated bycrossing mice homozygous for the loxP flanked stop cassette and Thy1-cremice expressing cre in the neurons of the postnatal cortex andhippocampus. Controls can include wild type mice with the same geneticbackground as the GILZ-Tg and Thy1-cre mice. The expression of GILZ mRNAand protein in the CNS can be determined. (See quotation from vendor andCo-I support letter for Thy1-Cre).

Assessment of the GILZ-Tg mice: The phenotype of GILZ-Tg mice can berecorded with respect to body weight, length and physical appearance.Brain tissue can be assessed for gain of GILZ protein byimmunohistochemistry and Western blot and by quantitative-PCR for GILZmRNA. In addition, the neuronal structure of the brain in the GILZ-Tgmice can be assessed by Nissl staining followed by immunohistochemicalstaining for neurons (Neu N), microglia (Iba1) and astrocytes (GFAP).

Cellular assays: Single cell suspensions from the brain homogenates ofwild type and GILZ-Tg mice can be processed to separate neuronal andglial cells by gradient centrifugation. Neuronal cell cultures can beexposed to Dex (1 μM) or Aβ₁₋₄₂ (10 μM). Cells harvested at the end of24 h and 48 h can be assessed for inflammatory markers (NF-κB,cytokines), anti-apoptosis (Bcl2, Bcl_(XL)) and pro-apoptotic (caspases,Bax, Bak) protein and gene expressions by Western blot, flow cytometryand RT-PCR.

Expected results, pitfalls and alternative measures: The use ofCre-based technology is an incredibly important tool to investigate bodysystems in health and disease. Although Thy1-cre is expressed robustlythroughout postnatal brain it is also observed in additional tissues. Todifferentiate real effects from artifacts and avoid misinterpretation ofresults, the following controls can be included: a) Cre backgroundstrains (C57BL/6), floxed controls, wild type controls and Thy1-Crecontrols. Alternatively, the effects of GILZ can be assessed byintroducing TAT-GILZ recombinant protein incrementally into wild typeneuronal cells.

GILZ overexpression in macrophages or cardiomyocytes has been shown toenhance Bcl-_(XL), suppress caspase-8 and protected against apoptosis.It was observed that the GILZ expression was reduced in the brain ofmice induced neuroinflammation and the reduction correlated with theenhanced cytokines. The expressions of apoptosis markers can be higherin GILZ-Tg mice. As Aβ could potentiate GC mediated neurotoxicity andthe GILZ effect is unknown, to compensate for compounding factors, theresponse of wild type neuronal cells exposed to high CORT will beanalyzed.

Example 23 Investigation of the Role of GILZ in AD

Evidence suggests that the inflammation aggravates neurotoxicity andaccelerates neurodegeneration in AD. NF-κB is a critical transcriptionfactor that regulates inflammatory responses, Aβ generation and taudeposition. Mechanistically, GILZ exerts anti-inflammatory andanti-proliferative effects by sequestering activated p65, thefunctionally critical subunit of NF-κB.

Overexpression of GILZ can Suppress Neuroinflammation and PreventNeurodegeneration:

Selection of AD model: To address whether GILZ is mechanisticallyinvolved in the pathogenesis of AD, 5×FAD mice that overexpress GILZ inthe nervous tissue can be generated. The 5×FAD is a rapid onset mousemodel of AD that bears 5 mutations linked to AD and recapitulates mainfeatures of AD. In this model, Aβ deposition and inflammation areobserved as early as 6 weeks. FIG. 22A-22D show elevated phosphorylatedNF-κB p65 correlating with gliosis and cytokines in the hippocampus andthe cortex of young 5×FAD mice.

Animals: Breeding colonies of 5×FAD can be maintained in transgenicanimal housing conditions in a 12-hour dark light cycle with access tofood and water. Non-transgenic wild type littermates can serve ascontrols. Hemizygous 5×FAD mice can be crossbred to the conditionalGILZ-Tg mice. Genotyping can be performed by PCR analysis of tail DNA.All experiments can be done blind with respect to the genotype of mice.The following groups of mice can be included: 1) experimental group ofGILZ-Tg 5×FAD mice; control groups can include 2) 5×FAD, 3) wild typeC57BL/6, 4) wild type, Thy1-Cre and 5) 5×FAD, Thy1-Cre.

All mice can be assessed for spatial learning and memory by Novel objectrecognition test and Morris water Maze at 4 weeks of age and thensacrificed for assessing CNS pathology, inflammation, Aβ load, gliosisand neuronal apoptosis by immunohistochemical, biochemical and molecularanalyses.

Suppression of neuroinflammation by a GILZ mimetic prevented decrease inGILZ expression in the hippocampus of 5×FAD mice (see FIGS. 22A-22D).Pertinently, GILZ has been shown to exert inhibitory effects onendothelial cell adhesive function and potentially inhibit leukocyterecruitment. Since neuroinflammation occur early in 5×FAD mice, the GILZoverexpression (FIG. 22A-22D) reduce Aβ load and suppress NF-κBactivation, inflammatory cytokines, PI3K signaling and prevent neuronalapoptosis.

Example 24 Decrease of Cytokines in Spinal Cord and Brain

Following spinal cord injury (SCI), inflammatory responses occur notonly in the spinal cord, but also in the brain, as demonstrated byincreased microglial activation (see FIG. 23A-23B and FIG. 24A-24B). AsHMGB1 is upregulated by SCI and secreted by microglia, lymphocytes,macrophages and other cells, and HMGB1 is known to be associated withdevelopment of neurodegenerative diseases like Alzheimer's disease, webelieve injured spinal cord-derived HMGB1 can elevate hippocampal andcortical microglial activation, which is associated with Alzheimer'sdisease among other neurodegenerative disorders.

Seven days following thoracic level 10 compression SCI, mice weresacrificed by approved methods and ˜4 mm spinal cord lesion area andtotal brain tissue were homogenized in RIPA lysis buffer andinflammatory cytokine levels for IFN-γ and IL-17 were upregulated atthis time point (blue bars). GILZ analog peptide given intraperitoneallyon day 6 post-SCI reduced these levels in brain, and partially in theinjured cord tissue (right bars) (FIG. 23A-23B). At this same time pointfollowing compression SCI, histological analysis of the hippocampus(FIG. 24A) and lesion center of the spinal cord (FIG. 24B) showprominent Iba-1+ activated microglial expression.

Example 25 Mechanisms by which the Immune-Inflammatory Responses InducedPost-SCI Increase the Risk for Neuroinflammation in the Brain

Preliminary data indicate hippocampus microglial activity is upregulatedfollowing SCI, and other reports show that HMGB1 is upregulated in theinjured cord following SCI. Thus, elevated HMGB1 protein in acuteinjured spinal cord tissue homogenate can activate reactivity and inducepro-inflammatory responses in hippocampal and cortical microglia.

Isolation of microglia from adult mouse brain: Microglia can be isolatesfrom the hippocampus and cortex of 6 adult C57BL/6 mice (5 weeks old).In brief, adult mice can be euthanized via approved methods, and thebrains removed, and the fresh hippocampus and cortex tissue quicklydissected and enzymatically digested. The resulting digested tissue canbe triturated and washed in Hank's balanced salt solution (HBSS) andcentrifuged to form a pellet. The pellet can be resuspended in culturemedium (DMEM/F12+10% fetal bovine serum [FBS]+1%penicillin/streptomycin) and washed again. Next the pellet can becentrifuged in a 30-70% Percoll gradient, and the microglia (visible atthe 70-37% gradient interphase) will be collected and washed with HBSS.The microglia can be counted and seeded in culture medium for furtherexperimentation.

Spinal cord injury: 8 week old female C57BL/6 mice can be randomlyassigned to four groups to produce a time course of injured spinal cordtissue homogenate to treat isolated and cultured adult mouse brainmicroglia: 1) sham surgery group (no SCI), 2) 1 d post-SCI, 3) 3dpost-SCI, 4) 7d and 28d post-SCI (n=4/group=20 mice). To produce SCI inthe mice, surgery and clip compression SCI can be performed. In brief,mice can be anesthetized with isoflurane, and the paraspinal musclesalong the T8-T11 vertebrae can be carefully dissected and the T10vertebra exposed. A laminectomy of the dorsal portion of the T10vertebrae exposing the spinal cord can be performed, and a 30 g forcevascular clip can be placed on the sides of the cord and carefullyclosed for compression. The cord can remain compressed for 1 minute,which will produce a moderate SCI.

The clip can then be removed, and the injured cord observed under thesurgical microscope for confirmation of proper injury. A band ofbruising and hemorrhage of blood in the cord can be visible at thelocation of clip compression. The muscle can be closed with sterilenylon suture, followed by the skin. The surgical site can be treatedwith antibacterial ointment and the mice placed in a clean home cage ona warming pad set to 37° C. to recover. The mice quickly recover fromisoflurane anesthesia and bilateral paralysis of the hindlimbs can beobserved as further confirmation of successful SCI. Mice in the shamsurgery group can undergo all steps of the surgery except spinal cordcompression.

Spinal cord isolation and homogenate preparation: Mice can be sacrificedin each group at the designated time points and transcardially perfusedwith normal saline. A 5 mm region of spinal cord centered over theinjury site can be rapidly dissected and minced in RIPA buffer aspreviously described. Tissue in RIPA buffer can then be homogenized witha dounce homogenizer, the tubes centrifuged to pellet debris, and thesupernatant homogenate isolated for treating microglial cultures.

HMGB1 ELISA: To test the concentration of HMGB1 from the homogenates inour SCI model, protein samples from the sham, 1d, 3d, 7d and 28d groupscan be assayed using a sandwich enzyme-linked immunosorbent assay(ELISA) for mouse HMGB1 (LifeSciences Biospan, Inc.), and absorbanceread on a plate reader (BioTek, Inc.) and concentrations determinedagainst a standard control curve. Purified recombinant mouse HMGB1protein (Biolegend, Inc.) can be assayed as a positive control.

Microglial treatment with spinal cord homogenates: Isolated microgliacan be seeded in 12-well culture dishes at a density of 2.5×10⁵ cells/mlin minimal essential medium (MEM) supplemented with 1% fetal bovineserum (FBS) and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml) andgrown to ˜80% confluence. HMGB1 is known to increase within hours in theinjured spinal cord. To test the effects of SCI homogenates onmicroglial activation and response, the experiment as described in Table3 will be performed.

TABLE 3 Experimental Design SC Homogenates (days post-SCI) Hippocampaland cortical Sham 1 3 7 28 microglial isolation Number of mice 4 4 4 4 46 Microglial Treatment Groups Normal culture medium only 1 μg/mllipopolysaccharide (LPS)  1% SC homogenate ✓ ✓ ✓ ✓ 10% SC homogenate ✓ ✓✓ ✓ 20% SC homogenate ✓ ✓ ✓ ✓ 3 μM HMGB1 w/o neutralizing antibody 3 μMHMGB1 w/neutralizing antibody SC = Spinal Cord

Three concentrations of homogenates in culture medium (1%, 10% and 20%vol/vol) can be applied to microglial cells and incubated at 37° C. and5% CO₂ for 12 h or 24 h The concentrations are extrapolated fromprevious studies suggesting that 0.3 μM of HMBG1 inhibiting Aβphagocytosis by microglia. Normal culture medium only can serve as anegative control and 1 μg/ml lipopolysaccharide (LPS), a knownstimulator of microglial activity, in normal culture medium will serveas a positive control. Cultures treated with known concentrations ofHMBG-1 (1-3 μM) in the presence or absence of monoclonal antibodyagainst HMBG1 can be included as a means of confirmation of the role ofHMBG1 in SCI homogenate.

Assessment of Microglial Responses:

Immunocytochemistry: Twelve hours after culture, microglia can be gentlyrinsed with PBS, and fixed with 4% paraformaldehyde. Cells can then beimmunostained for TLR-4, the chemokine ligand CCL12, the costimulatoryreceptors CD80 and CD86 and nuclear NF-κB p65. Furthermore, cells can beincubated with Hoechst 33342 to visualize microglial nuclei. Labeledfluorescence can be detected using a laser scanning confocal microscopeLSM 510 (Carl Zeiss, Jena, Germany).

ELISA for cytokines: The concentrations of inflammatory cytokines IL-6and TNF-α in culture supernatants harvested at the end of 12 h, 24 h and48 h can be assessed using appropriate OptEIA kits (BD Biosciences).

Quantitative RT-PCR (qRT-PCR): Total RNA isolated from culturedmicroglia can be reverse transcribed and qRT-PCR will be performed inABI 7000 system using the Superscript III Platinum SYBR Green One-StepqRT-PCR kit (Invitrogen), as described.

Primers: TLR4: (FP 5′-GCTTTCACCTCTGCCTTCAC-3′ (SEQ ID NO: 53);RP 5′-GA AACTGCCATGTTTGAGCA-3′ (SEQ ID NO: 54), IL-6:(FP 5′-AGTTGCCTTCTTGGGACTGA-3′ (SEQ ID NO: 55);RP 5′-TCCACGATTTCCCAGAGAAC-3′ (SEQ ID NO: 56), and RPS3:(FP 5′AATGAAC CGAAGCACACCATA-3′ (SEQ ID NO: 57);RP 5′-ATCAGAGAGTTGACCGCAGTT-3′) (SEQ ID NO: 58).

Product specificity can be confirmed by melting curve analysis andvisualization of single, appropriately sized band on a 2% agarose gel.Gene expression levels can be quantified using a cDNA standard curve anddata were normalized to RPS3, a housekeeping gene that was unaffected bytraumatic injury. Ability of HMGB1 primed microglia to respond to Aβ₁₋₄₂peptide:

Cultures of microglia exposed to spinal cord homogenates for 24 h asabove, can be rested in serum free media for 24 h. The cells can then bewashed in fresh culture medium and exposed to Aβ₁₋₄₂ at a finalconcentration of 10 μM/well and incubated for 4 h or 48 h. Cells andconditioned media will be harvested and stored for further analysis.Relative ATP concentration can be measured using the CTG kit (Promega)as described. Conditioned media collected can be assessed for specificcytokines using the OptEIA kits (BD Biosciences). The cell surfaceexpression of activation markers including MHC Class II, CD80 and CD 86can be assessed by immunocytochemistry and flow cytometry.

The concentration of HMBG1 in the spinal cord homogenates has been shownto be elevated within few hours after acute-SCI in both humans andanimal models. Furthermore, the level remains high for extended periodsin chronic SCI. The hippocampal microglia have been shown to expresselevated CCL12 in mice day 12 post-SCI. HMBG1 has been shown to activatemicroglia via TLR-4/NF-κB pathway. Hence, the TLR-4, CCL12 and nuclearp65 can be elevated in microglia exposed to spinal cord homogenatesobtained day 3-14 post-SCI. Previously, HMGB1 exposure has been shown toprime hippocampal microglial response to LPS. Since Aβ reactive T cellshave been shown to increase with aging, increase can be due to microgliafunction as efficient Aβ presenting cells. The SCI homogenate primedhippocampal and cortical microglia can exhibit activated phenotype.

Example 26 Investigation of Suppression of Activated NF-κB MediatedInflammation Following SCI as a Measure to Prevent HippocampalMicroglial Activation

The NF-κB pathway is important for the regulation of inflammation andapoptosis following SCI. The clinical benefits of the commonlyprescribed high-dose methylprednisolone steroid in the management ofSCI, is compromised by adverse effects. Glucocorticoid induced leucinezipper (GILZ) is a recently identified protein that mimics only thebeneficial effects of steroids and act by inhibiting the NF-κB p65protein. GILZ overexpression has been shown to suppress tissue damage inSCI. The suppression of NF-κB p65 can inhibit gliosis at the injury sitein SCI and prevent activation of hippocampal microgliosis.

This Example evaluates the efficacy of peptide analogs of GILZ (GA) ininhibiting microgliosis at injury sites and in the hippocampus of micesubjected to SCI (see Table 4). Adult C57BL/6 mice (Jackson Laboratory,Bar Harbor, Me.) can be administered lipopolysaccharide (LPS) (SigmaAldrich, Mo.) intraperitoneally at 250 mg/kg for 6 days. Serial 5μ thickcoronal sections of brain from naïve and LPS treated mice can beimmunostained for GILZ using TSC22D3 mAb (Catalog #: H00001831, Abnova,Walnut, Calif.). The peroxidase conjugated streptavidin biotin methodcan be used for detection and brown staining was considered positive.Staining specificity was confirmed by incubation with secondary antibodyalone. RNA isolated from brain homogenates was reverse transcribed.

TABLE 4 Experimental Design Biochemical Analysis Histologic Analysis n =6/group = 36 mice n = 6/group = 36 mice Groups Analyses: CytokineAnalyses: Brain and spinal Shamsurgery ELISA cord microglial stereologic7 d SCI + saline quantification and 7 d SCI + GA1 immunofluorescence 7 dSCI + GA2 labeling 7 d SCI + control peptide 7 d SCI + TAT carrier GA =GILZ analog, SCI = spinal cord injury

Spinal cord injury and treatment: SCI surgery can be performed asdescribed previously. To test the effect of GA peptides on CNSmicrogliosis post-SCI, C57BL/6 mice can be randomly assigned to sixgroups: 1) sham surgery, 2) SCI+saline, 3) SCI+GA peptide 1, 4) SCI+GApeptide 2, 5) SCI+non-specific peptide (control) and 6) SCI+carrier tag(GAG-TAT) injection (control) (n=12 per group [6 for biochemistry, 6 forhistologic analysis]×6 groups=72 mice).

GA preparation and administration: GA can be synthesized as peptideamides with amino terminal acetylation. The GA can be covalentlysynthesized with a cell penetrating peptide for intracellular delivery.Based on our studies in the AD model, GA1 (19 mM) and GA 2 (22 mM) canbe administered intraperitoneally on days 1 and 7 post SCI. Braintissues harvested at each time-point and end of the study can beevaluated for inflammation and neuronal loss/protection. Control groupscan include untreated SCI and scrambled peptide treated mice.

Protein preparation for biochemical analysis: After 7 days post-surgery,6 mice per group designated for biochemical analysis of NF-κB-mediatedinflammation can be transcardially perfused with saline and brain andspinal cord tissue isolated and prepared as described previously. Inbrief, the brain can be dissected and the cortex and hippocampus removedand placed in RIPA lysis buffer, and homogenized with a douncehomogenizer. The lysed tissue can be centrifuged and the supernatantcollected. The protein concentration of the supernatant can bedetermined with bichincionic acid assay (BCA, Pierce).

ELISA for cytokine expression analysis: Spinal cord and brainhomogenates can be assessed for cytokines (TNF-α, IL-12, IFN-g) andHMBG1 as described above.

Histologic assessment of brain and spinal cord microgliosis: Theremaining 6 mice can be euthanized on day 7 post-surgery, transcardiallyperfused with saline followed by perfusion with 4% paraformaldehyde(PFA) for fixation and histological assessment of microglial activationin the brain and spinal cord as described previously. Briefly, perfusedbrain and spinal cord tissue can be dissected and post-fixed in 4% PFAfor 24 hours. Then, the tissue can be placed in a 30% sucrose in 0.1 Mphosphate buffered saline (PBS) for cryopreservation, and then embeddedin Optimal Cutting Temperature medium for subsequent cryostatsectioning. Tissue can be serially sectioned at 20 um and mounted onSuperfrost Plus slides (Fisher Scientific). Immunofluorescence labelingwill be performed as previously described. Briefly, brain and spinalcord sections can be immunolabeled for microglia with rabbit anti-Iba1(1:500; Wako) overnight at 4° C., and the following day, the sectionswill be incubated with fluorophore-conjugated goat anti-rabbit secondaryantibody (1:200; Jackson ImmunoResearch Lab). Sections will then beincubated with Hoechst 33342 (1:500, Sigma-Aldrich) in PBS for nuclearlabeling, washed and coverslip mounted with Fluoromount G. In adjacentslides in the series, immunohistochemistry for Iba1 (1:500; Abcam, Inc.)can also be performed for unbiased stereologic quantification ofreactive microglia using a Stereolnvestigator system (Microbrightfield,Inc.) as previously described. Pre-immune serum can be used as a controlto confirm antibody specificity. Images can be obtained with anepifluorescent microscope (Nikon) and microglial reactivity can beassessed as a percentage of fluorescent labeling area per unit tissuearea.

Expected results: We have shown that both GA1 and GA2 suppressed gliosisand proinflammatory cytokines in the brain homogenates of mice subjectedto lipopolysaccharide induced neuroinflammation. Treatment with GA1 orGA2 can suppress inflammation at site of injury and this will bereflected in a decrease in the number of microglia in the hippocampus.

1.-20. (canceled)
 21. A method of treating a neurodegenerative diseasein a patient, the method comprising the step of administering to thepatient a composition comprising: a polypeptide from 8 to 12 amino acidresidues, the polypeptide comprising the sequence of XPXXP, wherein P isproline; and the first X is A or K, and the third X is Q, A, or S,wherein the polypeptide forms a PP_(II) helical conformation andinteracts with p65 residues F⁵³⁴ and F⁵⁴².
 22. The method of claim 21,wherein the polypeptide comprises the sequence of APKPYQPRG (SEQ ID NO:29).
 23. The method of claim 21, wherein the polypeptide comprises thesequence of EPAPXXPXX (SEQ ID NO: 1), and wherein the polypeptidecomprises a sequence selected from the group consisting of EPAPLAPYG(SEQ ID NO: 13), EPAPYQPEG (SEQ ID NO: 24), EPAPESPQV (SEQ ID NO: 17),and EPAPEQPDG (SEQ ID NO: 18).
 24. The method of claim 21, wherein thepolypeptide comprises the sequence of EPAPLAPYG (SEQ ID NO: 13).
 25. Themethod of claim 21, wherein the polypeptide comprises the sequence ofEPAPYQPEG (SEQ ID NO: 24).
 26. The method of claim 21, wherein thepolypeptide comprises the sequence of EPAPESPQV (SEQ ID NO: 17).
 27. Themethod of claim 21, wherein the polypeptide comprises the sequence ofEPAPEQPDG (SEQ ID NO: 18).
 28. The method of claim 21, wherein theneurodegenerative disease is Alzheimer's Disease.
 29. The method ofclaim 21, wherein the neurodegenerative disease is Parkinson's Disease.30. The method of claim 21, wherein the neurodegenerative disease ismultiple sclerosis.
 31. The method of claim 21, wherein theneurodegenerative disease is amyotrophic lateral sclerosis (ALS). 32.The method of claim 21, wherein the first X is A.
 33. The method ofclaim 21, wherein the first X is K.
 34. The method of claim 21, whereinthe third X is Q.
 35. The method of claim 21, wherein the third X is S.36. The method of claim 1, wherein the first X is A, and the third X isQ, S, or A.
 37. The method of claim 21, wherein the first X is A, andthe third X is Q.
 38. The method of claim 21, wherein the first X is A,and the third X is S.
 39. The method of claim 21, wherein the first X isA, and the third X is A.
 40. The method of claim 21, wherein the first Xis K, and the third X is Q.